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1

Fluorescence In Situ Hybridization  

Microsoft Academic Search

Dr. Seuss’s eloquent “One FISH, two FISH, red FISH, blue FISH” (1) could have been describing one of the most significant advancements in clinical cytogenetics, fluorescence in situ hybridization (FISH). The process, as described by Pinkel et al. in 1988 (2), involved fluorescent detection of probe DNA hybridized to chromosomal target sequences. The overall hybridization was essentially\\u000a the same one

Daynna J. Wolff; Stuart Schwartz

2

Molecular cytogenetics using fluorescence in situ hybridization.  

National Technical Information Service (NTIS)

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...

J. W. Gray W. L. Kuo J. Lucas D. Pinkel H. U. Weier

1990-01-01

3

Molecular cytogenetics using fluorescence in situ hybridization  

SciTech Connect

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.

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

1990-12-07

4

Sensitivity enhancement of fluorescence in situ hybridization on plant chromosomes  

Microsoft Academic Search

An improvedin situ hybridization procedure is presented, based on synchronization of root meristems of barley and wheat, enzymatic digestion, a protoplast drop technique, and the use of the fluorescent dye Cy3. The combination of these approaches resulted in a significant increase of well-spread metaphases suitable forin situ hybridization as compared to squash preparations, and to a significantly enhanced number and

W. Busch; R. Martin; R. G. Herrmann

1994-01-01

5

Concomitant Oncoprotein Detection with Fluorescence in Situ Hybridization (CODFISH)  

PubMed Central

We sought the validation of a three-color fluorescence-based system that simultaneously profiles Her-2/neu oncogene copy by fluorescence in situ hybridization (FISH) and Her-2/neu encoded protein by the use of a versatile alkaline phosphatase chromogen fast red K in either fluorescence or bright-field mode. Nuclei were counterstained with DAPI. Nineteen infiltrating ductal carcinomas of breast were comprehensively evaluated for Her-2/neu amplification/overexpression by direct and indirect FISH using digoxigenin (DigFISH) and direct fluorescently labeled probes, autoradiographic RNA:RNA in situ hybridization, and immunohistochemistry using monoclonal antibody CB11. CODFISH results correlated well with DigFISH, direct-label FISH, mRNA expression, and oncoprotein expression as assessed with CB11, and enabled simultaneous visualization of gene copy and protein. In addition, qualitative immunohistochemistry may be followed by CODFISH gene copy enumeration to clarify ambiguous cases.

Tubbs, Raymond R.; Pettay, James; Roche, Pat; Stoler, Mark H.; Jenkins, Robert; Myles, Jon; Grogan, Thomas

2000-01-01

6

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization  

Microsoft Academic Search

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial

Sven Poppert; Andreas Essig; Reinhard Marre; Michael Wagner; Matthias Horn

2002-01-01

7

Supernumerary ring chromosome 17 identified by fluorescent in situ hybridization  

SciTech Connect

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.

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

8

FISH-ing for Genes: Modeling Fluorescence "in situ" Hybridization  

ERIC Educational Resources Information Center

Teaching methods of genetic analysis such as fluorescence in situ hybridization (FISH) can be an important part of instructional units in biology, microbiology, and biotechnology. Experience, however, indicates that these topics are difficult for many students. The authors of this article describe how they created an activity that effectively…

Baker, William P.; Jones, Carleton Buck

2006-01-01

9

DNA sequence mapping by fluorescence in situ hybridization  

SciTech Connect

Various types of DNA probes, such as total genomic DNA, repetitive sequences, unique sequences, and composites of chromosome-specific DNA probes, can be used with fluorescence in situ hybridization (FISH) techniques to address research questions having to do with localization, mapping, and distribution of DNA in situ. FISH involves the formation of a heteroduplex between such DNA probes and chromatin targets on a microscope slide, which can be visualized with fluorescent reporter molecules. Three chromatin targets - metaphase chromosomes, somatic interphases, and zygote interphases - offer increasingly extended states of chromatin which can be strategically selected, individually or in combination, to address specific research questions of interest.

Brandriff, B.F.; Gordon, L.A.; Trask, B.J. (Univ. of California, Livermore (United States))

1991-01-01

10

RNA visualization in bacteria by fluorescence in situ hybridization.  

PubMed

Detecting localized RNA in bacteria is difficult due to the properties of RNA and the small size of the cell. Fluorescence in situ hybridization (FISH) has been an invaluable method for detecting and imaging RNA. In FISH, RNA is fixed in its native subcellular position through chemical cross-linking. An oligonucleotide probe conjugated to a fluorophore is annealed to the target RNA, and the target RNA/probe hybrid is visualized using fluorescence microscopy. This chapter describes the use of FISH to visualize tmRNA, a regulatory RNA required for trans-translation. The method can be adapted to visualize the localization of other regulatory and messenger RNAs as well. PMID:22736000

Russell, Jay H; Keiler, Kenneth C

2012-01-01

11

Detection of nitrifying bacteria in activated sludge by fluorescent in situ hybridization and fluorescence spectrometry  

Microsoft Academic Search

16S rRNA-targeted oligonucleotide probes for eubacteria (EUB338), ammonium-oxidizing bacteria (Nsm156) and nitrite-oxidizing bacteria (Nb1000) were used for the rapid detection of nitrifying bacteria in the activated sludge of a pilot nitrifying reactor by whole-cell, fluorescent in situ hybridization (FISH). Emission scanning and synchronous scanning fluorescence spectrometry were used to measure the hybridization. The binding of the probes at a temperature

In S. Kim; Volodymyr N. Ivanov

2000-01-01

12

Double fluorescence in situ hybridization in fresh brain sections.  

PubMed

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure. PMID:20736918

Jeong, Jin Kwon; Chen, Zhuoxun; Tremere, Liisa A; Pinaud, Raphael

2010-01-01

13

Double Fluorescence in situ Hybridization in Fresh Brain Sections  

PubMed Central

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Jeong, Jin Kwon; Chen, Zhuoxun; Tremere, Liisa A.; Pinaud, Raphael

2010-01-01

14

Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization  

Microsoft Academic Search

Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully

Masatoki Taga; Minoru Murata

1994-01-01

15

Spatial Distribution of Sperm-Derived Chromatin in Zygotes Determined by Fluorescence In situ Hybridization.  

National Technical Information Service (NTIS)

Fluorescence in situ hybridization was used to determine the spatial distribution of chromatin in zygote pronuclei. A hybrid system involving golden hamster eggs and individual human sperm permitted use of DNA probes specific for the entire human chromoso...

B. F. Brandriff L. A. Gordon

1992-01-01

16

Human cDNA mapping using fluorescence in situ hybridization  

SciTech Connect

Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

Korenberg, J.R.

1993-03-04

17

Sexing of Dog Sperm by Fluorescence In Situ Hybridization  

PubMed Central

Abstract Effective preselection of sex has been accomplished in several species of livestock and also in humans using the flow cytometric sperm sorting method. A guaranteed high sorting accuracy is a key prerequisite for the widespread use of sperm sexing. The standard validation method is flow cytometric remeasurement of the DNA content of the sexed sperm. Since this method relies on the same instrument that produced the original sperm separation, it is not truly independent. Therefore, to be able to specifically produce either male or female offspring in the dog, we developed a method of direct visualization of sex chromosomes in a single sperm using fluorescence in situ hybridization (FISH) as a validation method. Denaturation of canine spermatozoa by immersion in 1 M NaOH for 4 min yielded consistent hybridization results with over 97% hybridization efficiency and a good preservation of sperm morphology. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X- and Y-chromosome-bearing spermatozoa in any of the three dogs. In addition, the mean purities of flow-sorted sex chromosomes in spermatozoa of the three dogs were 90.8% for the X chromosome fraction and 89.6% for the Y chromosome fraction. This sorting was evaluated by using the dual color FISH protocol. Therefore, our results demonstrated that the FISH protocol worked reliably for both unsorted and sexed sperm samples.

OI, Maya; YAMADA, Keisuke; HAYAKAWA, Hiroyuki; SUZUKI, Hiroshi

2012-01-01

18

The use of fluorescent in situ hybridization in male infertility  

PubMed Central

Male factors are implicated in up to 50% of couples being evaluated and treated for infertility with advanced assisted reproductive technologies. Genetic abnormalities, including sperm chromosome aneuploidy as well as structural aberrations, are one of the major causes of infertility. The use of chromosome-specific DNA probes labeled with fluorochromes, particularly the combination with multiple probes, has been used to indirectly study the sperm chromosome by fluorescent in situ hybridization (FISH). Clinically, this technique is also used to assess the sperm of men recovering from gonadotoxic treatment. Recent advances in this technology facilitate the evaluation of sperm aneuploidy. Sperm FISH is a widely used screening tool to aid in counseling couples with severe male factor infertility, especially in cases of prior repeated in vitro fertilization/intracytoplasmic sperm injection failure or recurrent pregnancy loss. Automation of FISH imaging and analysis, as well as the development of emerging techniques such as comparative genomic hybridization, will all contribute to the promise of future diagnostic approaches aimed at improving the quality, ease, and efficiency of aneuploidy analysis.

Hwang, Kathleen; Weedin, John W.; Lamb, Dolores J.

2010-01-01

19

Multiplex-fluorescence in situ hybridization for chromosome karyotyping.  

PubMed

Multiplex-fluorescence in situ hybridization (M-FISH) was initially developed to stain human chromosomes--the 22 autosomes and X and Y sex chromosomes--with uniquely distinctive colors to facilitate karyotyping. The characteristic spectral signatures of all different combinations of fluorochromes are determined by multichannel image-analysis methods. Advantages of M-FISH include rapid analysis of metaphase spreads, even in complex cases with multiple chromosomal rearrangements, and identification of marker chromosomes. The M-FISH technology has been extended to other species, such as the mouse. Furthermore, in addition to painting probes, the method has been used with a variety of region-specific probes. M-FISH has even recently been used for 3D studies to analyze the distribution of human chromosomes in intact and preserved interphase nuclei. Hence, M-FISH has evolved into an essential tool for both clinical diagnostics and basic research. In this protocol, we describe how to use M-FISH to karyotype chromosomes, a procedure that takes approximately 14 d if new M-FISH probes have to be generated and 3 d if the M-FISH probes are ready to use. PMID:17406400

Geigl, Jochen B; Uhrig, Sabine; Speicher, Michael R

2006-01-01

20

Characterization of Robertsonian translocations by using fluorescence in situ hybridization.  

PubMed

Fluorescence in situ hybridization with five biotin-labeled probes (three alphoid probes, a probe specific for beta-satellite sequences in all acrocentric chromosomes, and an rDNA probe) was used to characterize 30 different Robertsonian translocations, including three t(13;13); one t(15;15), four t(21;21), three t(13;14), two t(13;15), two (13;21), two t(13;22), one t(14;15), eight t(14;21), two t(14;22), and two t(21;22). Of 8 de novo homologous translocations, only one t(13;13) chromosome was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. The three monocentric nonhomologous translocations included both of the t(13;21) and one t(21;22). Two of 26 translocations studied using the beta-satellite probe showed a positive signal, while rDNA was undetectable in 10 cases studied. These results indicate that most homologous Robertsonian translocations appear monocentric, while the bulk of nonhomologous translocations show two alphoid signals. A majority of the breakpoints localized using this analysis seem to be distal to the centromere and just proximal to the beta-satellite and nuclear-organizing regions. PMID:1729886

Wolff, D J; Schwartz, S

1992-01-01

21

Detection of dengue group viruses by fluorescence in situ hybridization  

PubMed Central

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.

2012-01-01

22

Chromosome replicating timing combined with fluorescent in situ hybridization.  

PubMed

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation(1). Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of >3 hours that affects the entire chromosome(2,3). Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome(4). Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes(5). Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from(6). In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population. PMID:23271586

Smith, Leslie; Thayer, Mathew

2012-01-01

23

Fluorescent in situ hybridization on mitotic chromosomes of mosquitoes.  

PubMed

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions. Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti and Cx. quinquefasciatus, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti, the accumulation of multiple chromosomal rearrangements in cell line chromosomes makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups. PMID:23007640

Timoshevskiy, Vladimir A; Sharma, Atashi; Sharakhov, Igor V; Sharakhova, Maria V

2012-01-01

24

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes  

PubMed Central

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Timoshevskiy, Vladimir A.; Sharma, Atashi; Sharakhov, Igor V.; Sharakhova, Maria V.

2012-01-01

25

Multiple and sensitive fluorescence in situ hybridization with rhodamine-, fluorescein-, and coumarin-labeled DNAs  

Microsoft Academic Search

We have tested the use of several newly developed red, green, and blue fluorescent dUTPs in direct, multiple, and sensitive fluorescence in situ hybridization procedures. Among the ones tested, the tetramethylrhodamine-dUTP proved to give the best sensitivity; using conventional epifiuorescence microscopy, cosmids could be visualized with a hybridization efficiency of 90%. Fluorescein-dUTP permitted visual cosmid detection with 50% efficiency, and,

J. Wiegant; C. C. Wiesmeijer; J. M. N. Hoovers; E. Schuuring; A. d’Azzo; J. Vrolijk; H. J. Tanke; A. K. Raap

1993-01-01

26

Image analysis of cells stained by fluorescent in situ hybridization  

SciTech Connect

Specific nucleic acid sequences of about 1 kb can be detected on a routine basis using FISH methods. This is achieved by indirect techniques and conventional microscopy, or with fluorescent probes (direct methods) and integrating detection devices (e.g. CCD cameras). Labeling of probes with defined ratios of two or three fluorophores allows localization of multiple targets, especially when the signals are quantified by image analysis. This requires fluoroescence microscopes equipped with multi band pass filters and computer controlled filter wheels for sequential recording of red, green and blue images using a B/W CCD camera, and correction of occurring image shifts. This combined approach was applied to localize and map genes on chromosomes. In case of nuclei with long extentions of almost linear DNA molecules, (so-called halo preparations), single hybridization sides could be shown.

Tanke, H.J.; Raap, A.K.; Wiegant, J.; Vrolijk, J. (Leiden Univ. (Netherlands))

1993-01-01

27

Faster identification of pathogens in positive blood cultures by fluorescence in situ hybridization in routine practice  

Microsoft Academic Search

Rapid identification of microorganisms in blood cultures is required to optimize empirical treatment at an early stage. Fluorescence in situ hybridization (FISH) can reduce the time to identification of microorganisms in growth-positive blood cultures. In this study, we evaluated the performance, time to identification, and potential clinical benefits of FISH compared to those of conventional culture methods in routine practice.

Remco P. H. Peters; Paul H. M. Savelkoul; Alberdina M. Simoons-Smit; Sven A. Danner; Agtmael van M. A

2006-01-01

28

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

Microsoft Academic Search

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

Galina G Hovhannisyan

2010-01-01

29

Mechanistic Approach to the Problem of Hybridization Efficiency in Fluorescent In Situ Hybridization  

PubMed Central

In fluorescent in situ hybridization (FISH), the efficiency of hybridization between the DNA probe and the rRNA has been related to the accessibility of the rRNA when ribosome content and cell permeability are not limiting. Published rRNA accessibility maps show that probe brightness is sensitive to the organism being hybridized and the exact location of the target site and, hence, it is highly unpredictable based on accessibility only. In this study, a model of FISH based on the thermodynamics of nucleic acid hybridization was developed. The model provides a mechanistic approach to calculate the affinity of the probe to the target site, which is defined as the overall Gibbs free energy change (?G°overall) for a reaction scheme involving the DNA-rRNA and intramolecular DNA and rRNA interactions that take place during FISH. Probe data sets for the published accessibility maps and experiments targeting localized regions in the 16S rRNA of Escherichia coli were used to demonstrate that ?G°overall is a strong predictor of hybridization efficiency and superior to conventional estimates based on the dissociation temperature of the DNA/rRNA duplex. The use of the proposed model also allowed the development of mechanistic approaches to increase probe brightness, even in seemingly inaccessible regions of the 16S rRNA. Finally, a threshold ?G°overall of ?13.0 kcal/mol was proposed as a goal in the design of FISH probes to maximize hybridization efficiency without compromising specificity.

Yilmaz, L. Safak; Noguera, Daniel R.

2004-01-01

30

Rapid Prenatal Diagnosis of Aneuploidies in Uncultured Amniocytes by Fluorescence in situ Hybridization  

Microsoft Academic Search

Objective: A new method in prenatal diagnostics allows to demonstrate certain numeric chromosomal aneuploidies in amniotic cells within 24 h in contrast to conventional methods which take 1–3 weeks. Materials: The experience with this rapid fluorescence in situ hybridization (FISH) method is compared to standard karyotyping and its clinical relevance is described in a large clinical pilot study. FISH on

Bernd Eiben; Witold Trawicki; Wilhelm Hammans; Richard Goebel; Michael Pruggmayer; Jörg T. Epplen

1999-01-01

31

Genomic aberrations in plasma cell leukemia shown by interphase fluorescence in situ hybridization  

Microsoft Academic Search

A combination of cytoplasmic immunofluorescence to detect the immunoglobulin light chain and an interphase fluorescence in situ hybridization technique was used to study the recurrent genetic abnormalities in 14 patients with plasma cell leukemia (PCL). Of the 14 patients studied, 5 had primary and 9 secondary PCL. Chromosomal abnormalities were detected in all 14 patients (100%). Deletions of 13q14 were

Hong Chang; Stephen Sloan; Dan Li; Bruce Patterson

2005-01-01

32

Presence of endophytic bacteria in Vitis vinifera leaves as detected by fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) in combination with confocal laser scanning microscopy (CLSM) was applied to detect\\u000a and localize bacterial colonies in leaf tissues of Vitis vinifera. Leaves were cleared to minimize the autofluorescence of plant fragments. The use of fluorescently labeled bacterial probe\\u000a EUB338 on discolored grapevine leaf disks allowed the estimation of the spatial distribution of different bacterial

Sandra Lo Piccolo; Valeria Ferraro; Antonio Alfonzo; Luca Settanni; Danilo Ercolini; Santella Burruano; Giancarlo Moschetti

2010-01-01

33

Fluorochrome and Fluorescent In Situ Hybridization to Monitor Bioaerosols in Swine Buildings  

Microsoft Academic Search

Total microbial cell concentration, viability, and culturability of bioaerosols in swine buildings were monitored by using epifluorescence microscopy with fluorochrome (EFM\\/FL) with four fluorescent dyes (AO, DAPI, PI, and YOPRO-1) and by using fluorescent in situ hybridization (FISH) with five oligonucleotide probes (fl-Univ, fl-EUB, cy-EUK, fl-PSMg, and fl-NotEUB) probes. Results from these two non-culture-based methods were then compared with those

Miao-Ching Chi; Chih-Shan Li

2005-01-01

34

Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization  

Microsoft Academic Search

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such

Ramon Rosselló-Mora; Natuschka Lee; Josefa Antón; Michael Wagner

2003-01-01

35

FISH - (Fluoresence In Situ Hybridization)  

NSDL National Science Digital Library

Fluorescence in situ hybridization (FISH) is a process which vividly paints chromosomes or portions of chromosomes with fluorescent molecules. This technique is useful for identifying chromosomal abnormalities and gene mapping.

BEGIN:VCARD VERSION:2.1 FN:Darryl Leja N:Leja;Darryl ORG:National Human Genome Research Institute REV:2005-04-04 END:VCARD

2005-04-04

36

CCD microscopy and image analysis of cells and chromosomes stained by fluorescence in situ hybridization  

Microsoft Academic Search

Summary  This paper reviews methods and applications of CCD microscopy for analysing cells and chromosomes subjected to fluorescence in situ hybridization (FISH). The current status of indirect and direct FISH staining methods with respect to probe labelling, detection sensitivity, multiplicity and DNA resolution is summarized. Microscope hardware, including special multi-band pass filters and CCD cameras required for FISH analysis, is described.

H. J. Tanke; R. J. Florijn; J. Wiegant; A. K. Raap; J. Vrolijk

1995-01-01

37

Mapping pachytene chromosomes of coffee using a modified protocol for fluorescence in situ hybridization.  

PubMed

Fluorescence in situ hybridization (FISH) is the most direct method for physically mapping DNA sequences on chromosomes. Fluorescence in situ hybridization mapping of meiotic chromosomes during the pachytene stage is an important tool in plant cytogenetics, because it provides high-resolution measurements of physical distances. Fluorescence in situ hybridization mapping of coffee pachytene chromosomes offers significant advantages compared with FISH mapping of somatic chromosomes, because pachytene chromosomes are 30 times longer and provide additional cytological markers. However, the application of this technique to pachytene chromosomes has been complicated by problems in making preparations of meiotic chromosomes and by difficulties in the application of standard FISH protocols. We have been able to overcome most of these obstacles in applying the FISH technique to the pachytene chromosomes of coffee plants. Digesting the external callose layer surrounding the pollen mother cells (PMCs) in conjunction with other procedures permitted suitable pachytene chromosomes to be obtained by increasing cell permeability, which allowed the probe sequences to enter the cells. For the first time, hybridization signals were registered on coffee pachytene chromosomes using the FISH technique with a repetitive sequence as a probe. We obtained slides on which 80 % of the PMCs had hybridization signals, resulting in FISH labelling with high efficiency. The procedure does not seem to be dependent on the genotype, because hybridization signals were detected in genetically different coffee plants. These findings enhance the possibilities for high-resolution physical mapping of coffee chromosomes. PMID:24244840

Iacia, Ana Amélia Sanchez; Pinto-Maglio, Cecília A F

2013-01-01

38

Microbial Community Composition of Wadden Sea Sediments as Revealed by Fluorescence In Situ Hybridization  

Microsoft Academic Search

The microbial community composition of Wadden Sea sediments of the German North Sea coast was investigated by in situ hybridization with group-specific fluorescently labeled, rRNA-targeted oligonucleotides. A large fraction (up to 73%) of the DAPI (4*,6-diamidino-2-phenylindole)-stained cells hybridized with the bacterial probes. Nearly 45% of the total cells could be further identified as belonging to known phyla. Members of the

ENRIC LLOBET-BROSSA; RAMON ROSSELLO ´-MORA; RUDOLF AMANN; Max Planck

1998-01-01

39

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)  

PubMed Central

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1. FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Vyboh, Kishanda; Ajamian, Lara; Mouland, Andrew J.

2012-01-01

40

Painting of parental chromatin in Beta hybrids by multi-colour fluorescent in situ hybridization.  

PubMed

Sugar beet (Beta vulgaris L.) is a relatively young crop and has a narrow gene pool. In order to introduce genetic variability into the crop, interspecific hybrids, selected from crosses with wild beets of the sections Corollinae and Procumbentes, have been generated. The introgressed B. procumbens chromatin carries resistance genes to beet cyst nematode Heterodera schachtii Schm. These lines are important for breeding of nematode-resistant sugar beet, while Corollinae species are potential donors of tolerance to biotic and abiotic stresses such as drought or saline soils. We have used in situ hybridization of genomic DNA to discriminate the parental chromosomes in these interspecific hybrids. Suppression of cross-hybridization by blocking DNA was not necessary indicating that the investigated Beta genomes contain sufficient species-specific DNA enabling the unequivocal determination of the genomic composition of the hybrids. Interspecific hybrid lines with an additional chromosome (2n = 18 + 1), chromosome fragment (2n = 18 + fragment) or translocation of B. procumbens (2n = 18) were analysed by genomic in situ hybridization (GISH) at mitosis and meiosis. Species-specific satellites and ribosomal genes used in combination with genomic DNA or in rehybridization experiments served as landmark probes for chromosome identification in hybrid genomes. The detection of a B. procumbens translocation of approx. I Mbp demonstrated the sensitivity and resolution of GISH and showed that this approach is a powerful method in genome analysis projects of the genus Beta. PMID:12099348

Desel, Christine; Jansen, Rita; Dedong, Gue; Schmidt, Thomas

2002-02-01

41

Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization  

Microsoft Academic Search

This study combines conventional cytogenetics, fluorescence in situ hybridization (FISH), multiplex-FISH and comparative genomic hybridization (CGH). In applying this multimodal approach on the human leukemia cell line K562, the chromosome composition was refined in detail and compared with data from the literature. A hypotriploid karyotype with a modal chromosome number of 67, and 21 unique marker chromosomes were identified. The

Sabine Naumann; Dirk Reutzel; Michael Speicher; Hans-Jochen Decker

2001-01-01

42

Comparative analysis of two thyroid tumor cell lines by fluorescence in situ hybridization and comparative genomic hybridization  

Microsoft Academic Search

Tumors and tumor-derived cell lines are typically chromosomally complex and heterogeneous. These features complicate the description of their karyotype. As a first approach to the chromosomal characterization of the two near-triploid thyroid tumor cell lines, BCPAP and FTC133, the techniques of fluorescence in situ hybridization and comparative genomic hybridization were used and compared. Most of the results obtained by the

Chiara Corso; Hacan Ulucan; Elizabeth M. Parry; James M. Parry

2002-01-01

43

Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization  

Microsoft Academic Search

The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic

Yoshiteru Aoi; Tomoko Miyoshi; Toshiyuki Okamoto; Satoshi Tsuneda; Akira Hirata; Atsushi Kitayama; Teruyuki Nagamune

2000-01-01

44

Cytogenetic and Fluorescence In Situ Hybridization Studies in Four Cases of Plasma Cell Leukemia  

Microsoft Academic Search

We present a cytogenetic and fluorescence in situ hybridization (FISH) study, using centromeric probes for chromosomes 3, 7, 11, and 18, TP53 gene (17p13), and RB-1 locus (13q14) DNA probes, in four cases of plasma cell leukemia (PCL). Among the four cases, three presented monosomy of the RB-1 locus and one monoallelic deletion of the TP53 gene. The present report

Elisabet Lloveras; Francesc Solé; Blanca Espinet; Carles Besses; Antoni Asensio; Eugčnia Abella; Soledad Woessner; Lourdes Florensa

2000-01-01

45

Detection of chromosome aberrations in interphase nuclei using fluorescence in situ hybridization technique.  

PubMed Central

We report here several experiences of interphase cytogenetics, using fluorescence in situ hybridization (FISH) technique, for the detection of chromosome aberrations. FISH, using alpha satellite specific probes of 18, X, Y chromosomes, was done in interphase nuclei from peripheral blood of patients with Edwards' syndrome, Klinefelter's syndrome and Turner's syndrome with healthy male and female controls, respectively. The distributions of fluorescent signals in 100 interphase nuclei were well correlated with metaphase findings. Nowadays FISH plays an increasingly important role in a variety of research areas, including cytogenetics, prenatal diagnosis, tumor biology, gene amplification and gene mapping.

Song, H. G.; Choi, S. O.; Kang, I. B.

1993-01-01

46

microFIND(®) approach to fluorescent in situ hybridization (FISH).  

PubMed

FISH technology has gained increasing attention in the management of cancer disease, either for predictive or prognostic indications. Molecular cytogenetics has greatly improved diagnostic capability of classical cytogenetics analysis of metaphase-based chromosome for the identification of genetic aberrations. The availability of a large number of fluorescent probes, each specific for different genetic lesions, together with a robust protocol for interphase FISH, provide the pathologist with the essential tools for an accurate evaluation of patient's disease. Hemato-oncological and many of the solid tumors have been comprehensively characterized by peculiar genetic defects and are now routinely evaluated by interphase FISH. Despite the reliability of the method, which has undergone only minor changes since the 1970s, FISH assay is still hampered by reagents cost, preventing its adoption in large-scale oncological screening. In this chapter we describe a major improvement of interphase FISH assay for cytological samples through the description of the miniaturized device microFIND(®) that offers, besides reduction of cost per assay, a completely novel vision to the FISH technology, thanks to the perspective of full automation of FISH assay using a dedicated robotic platform for microFIND(®) handling, (not presently described in the chapter). PMID:23329459

Zanardi, Andrea; Barborini, Emanuele; Carbone, Roberta

2013-01-01

47

Towards unlimited colors for fluorescence in-situ hybridization (FISH).  

PubMed

We describe a FISH protocol that allows rehybridization of complex DNA probes up to four times to the same specimen. This strategy, which we termed ReFISH, opens a wide range of new applications to conventional band pass filter epifluorescence microscopy. These include M-FISH karyotyping and cross-species color banding that emulate multiplex probe sets labeled with up to 12 fluorochromes in sequential hybridizations to the same specimen. We designed a human 24-color karyotyping probe set in combination with a 29-color cross-species color banding probe set using gibbon painting probes. Applying the ReFISH principle, 53 painting probes on individual metaphases were discriminated. This allowed simultaneous screening for inter- and intrachromosomal rearrangements on normal human diploid cells, a HeLa derived cell line, and highly rearranged gibbon chromosomes. Furthermore, the present ReFISH experiments successfully combine 24-color FISH with laser scanning confocal microscopy to study the 3D organization of all 46 human chromosome territories in individual interphase cell nuclei. PMID:12067211

Müller, Stefan; Neusser, Michaela; Wienberg, Johannes

2002-01-01

48

Bacterioplankton Compositions of Lakes and Oceans: a First Comparison Based on Fluorescence In Situ Hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific

FRANK OLIVER GLOCKNER; BERNHARD M. FUCHS; RUDOLF AMANN

1999-01-01

49

Localization of the B-hordein locus on barley chromosomes using fluorescence in situ hybridization  

Microsoft Academic Search

The B-hordein gene family consists of at least 13 genes that appear to be clustered in two regions on the barley chromosome 1H (5). Using fluorescence in situ hybridization techniques, we have localized the B-hordein locus (Hor2) to the distal end of the short arm of chromosome 1H (5), corresponding to the genetically determined position, 91% distal on the short

H. Lehfer; W. Busch; R. Martin; R. G. Herrmann

1993-01-01

50

Simultaneous Detection and Differentiation of Staphylococcus Speciesin Blood Cultures Using Fluorescence in situ Hybridization  

Microsoft Academic Search

Objective: To develop a new protocol for the simultaneous detection and differentiation of Staphylococcus aureus and coagulase-negative staphylococci (CoNS) in blood cultures using fluorescence in situ hybridization (FISH), without cultivation and biotyping. Materials and Methods: Oligonucleotide probes were used to target the variable regions of the 16S rRNA of S. aureus and CoNS, the probes were labeled with fluorochrome Cy3

Pei Wang

2010-01-01

51

Efficacy of two fluorescence in situ hybridization (FISH) probes for diagnosing malignant pleural effusions.  

PubMed

It is difficult to differentiate tumor cells in pleural fluid from reactive benign mesothelium. Fluorescence in situ hybridization (FISH) can increase diagnostic accuracy. Two hundred pleural fluid samples were analyzed by using FISH probes for chromosomes 11 and 17. Histological analysis was used to diagnose cancer. Clinical, radiological, and histological data were used to exclude malignancy. Eighty-two pleural effusion samples had positive cytology, 51 were benign, and 67 were atypical, but inconclusive. The 82 positive cases were confirmed to be malignant. Among the 51 negative cytology cases, videothoracoscopy-guided pleural biopsy revealed malignancy in three; aneuploid cells were detected by FISH in all cases. In 43 of the 67 cases with inconclusive cytology, malignancy was confirmed based on histology and fluorescence in situ hybridization. One case of parapneumonic effusion with no evidence of cancer during clinical follow-up had a suspicious cytology and positive fluorescence in situ hybridization result. The remaining 23 cases had no histological, radiological, clinical, or genetic evidence of malignancy. This study demonstrated that cytogenetic analysis of fresh pleural fluid samples using only two FISH probes is a valuable ancillary method for the identification of malignant pleural effusion, particularly in cases in which oncotic cytology is inconclusive. PMID:23453645

Rosolen, Débora C B; Kulikowski, Leslie D; Bottura, Giorgio; Nascimento, Amon M; Acencio, Milena; Teixeira, Lisete; Vargas, Francisco S; Sales, Roberta K; Antonangelo, Leila

2013-06-01

52

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis  

Microsoft Academic Search

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer

Trond Stokke; Colin Collins; Wen-Lin Kuo; David Kowbel; Farideh Shadravan; Minna Tanner; Anne Kallioniemi; Olli-P. Kallioniemi; Daniel Pinkel; Larry Deaven; Joe W. Gray

1995-01-01

53

Characterization of a Chromosomally Complex Lung Cancer Cell Line Using Multiwell Fluorescence In Situ Hybridization  

Microsoft Academic Search

The chromosomal characterization of a non-small cell lung cancer cell line (NCIH358) is described. This characterization was achieved using a simple, cheap and technically straightforward multiwell fluorescence in situ hybridization (FISH) method. The many and complex chromosome rearrangements identified by this method could not be defined using conventional G-banded chromosome analysis, and have not been previously described. For the detailed

C. Mackie Ogilvie; S Shemilt; A. F Davies; S Weber-Hall; C.-Y Chuang; V Sundaresan

2000-01-01

54

Sperm ldentification in Maize by Fluorescence in Situ Hybridization  

Microsoft Academic Search

The two sperm cells of common origin within the pollen tube of flowering plants are each involved in a fertilization event. It has long been recognized that preferential fusion of one sperm with the egg can occur in B chromosome-containing lines of maize. If the second pollen mitosis begins with a single B chromosome, nondisjunction will result in one sperm

Liang Shi; Tong Zhu; H. Lloyd Mogensen; Paul Keim

55

Actinobacillus pleuropneumoniae osteomyelitis in pigs demonstrated by fluorescent in situ hybridization.  

PubMed

Necrotizing osteomyelitis and fibrinopurulent arthritis with isolation of Actinobacillus pleuropneumoniae serotype 2 is reported in two pigs from a herd with lameness and mild coughing problems among 8 to 12-week-old pigs. Application of fluorescent in situ hybridization targeting 16S ribosomal RNA of A. pleuropneumoniae in formalin-fixed tissue was performed to verify the association of A. pleuropneumoniae with the bone and joint lesions. By in situ hybridization A. pleuropneumoniae was demonstrated as multiple microcolonies or single cells dispersed in focal fibrinonecrotizing pleuropneumonia, in joints with arthritis, and in bone necroses including lysis of growth plate and suppurative inflammation in the adjacent trabecular metaphysis, thus demonstrating that well-known infections manifest new, unusual lesions. PMID:10332836

Jensen, T K; Boye, M; Hagedorn-Olsen, T; Riising, H J; Angen, O

1999-05-01

56

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

SciTech Connect

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.

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

1990-08-01

57

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

PubMed

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. PMID:2201023

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

1990-08-01

58

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae  

PubMed Central

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

McIsaac, R. Scott; Silverman, Sanford J.; Parsons, Lance; Xu, Ping; Briehof, Ryan; McClean, Megan N.; Botstein, David

2013-01-01

59

Fluorescence in situ Hybridization Applied to Domestic Animal Cytogenetics  

Microsoft Academic Search

The aim of this article is not to present an exhaustive review of molecular cytogenetics applications in domestic animal species, but more to illustrate the considerable contribution of these approaches in diagnostics and research in economically important species. A short presentation of the main applications of molecular cytogenetics in humans points out the domains in which it has become an

J. Rubes; A. Pinton; A. Bonnet-Garnier; V. Fillon; P. Musilova; K. Michalova; S. Kubickova; A. Ducos; M. Yerle

2009-01-01

60

Construction of repeat-free fluorescence in situ hybridization probes.  

PubMed

FISH probes are generally made out of BAC clones with genomic DNA containing a variable amount of repetitive DNA that will need to be removed or blocked for FISH analysis. To generate repeat free (RF) Probes without loss in genomic coverage, a random library is made from BAC clones by whole-genome amplification (WGA). Libraries are denatured in the presence of excess C(0)t-1 DNA and allowed to re-anneal followed by digestion of all double-stranded elements by duplex-specific nuclease (DSN). Selective amplification of all elements not containing repetitive sequences is realized by a sequential amplification. The final RF products can be re-amplified and used as a stock for future probe production. The RF probes have a lower background, the signal intensity build up is faster and there is no need for blocking DNA. The signal to background ratio of the RF was higher as compared to repeat containing probes. PMID:22123742

Swennenhuis, Joost F; Foulk, Brad; Coumans, Frank A W; Terstappen, Leon W M M

2012-02-01

61

Detection of transgenes in three genetically modified rice lines by fluorescence in situ hybridization  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) using T-DNA probes was applied to localize transgenes onto specific chromosomes\\u000a and confirm the steady integration of transferred genes in three genetically modified (GM) rice lines, LS28 (event LS30-32-20-1),\\u000a Cry1Ac1 (event C7-1-9-1) and LS28×Cry1Ac1 (event L\\/C1-1-3-1), which are a rice leaf blast-resistant single trait GM line,\\u000a a leaf folder-resistant single trait GM line, and a

Hye Mi Park; Eun Jin Jeon; Nomar Espinosa Waminal; Kong Sik Shin; Soon Jong Kweon; Beom-Seok Park; Seok Cheol Suh; Hyun Hee Kim

2010-01-01

62

Three-dimensional acrylamide fluorescence in situ hybridization for plant cells.  

PubMed

Plant meiosis involves complex and dynamic processes that occur within the space inside the nucleus. Direct inspection of meiotic chromosomes by fluorescence microscopy has been used to investigate many of these processes. In particular, optical sectioning microscopy of fluorescence in situ hybridization (FISH)-stained nuclei provides three-dimensional spatial information about the organization and distribution of specific sequences and chromosomal loci within the nucleus. Here we provide a fully detailed three-dimensional (3D) acrylamide FISH method for the analysis of plant meiotic nuclei. Several examples illustrate the versatility of this technique for the investigation of meiotic telomere dynamics in maize, Arabidopsis, and oat. Additional examples of 3D FISH include chromosome painting in a maize chromosome-addition line of oat and telomere FISH with maize nuclei from plants expressing a fluorescently tagged fusion protein, histone H2B-mCherry. PMID:23559202

Howe, Elizabeth S; Murphy, Shaun P; Bass, Hank W

2013-01-01

63

Single-mRNA counting using fluorescent in situ hybridization in budding yeast  

PubMed Central

Fluorescent in situ hybridization (FISH) allows the quantification of single mRNAs in budding yeast using fluorescently labeled single-stranded DNA probes, a wide-field epifluorescence microscope and a spot-detection algorithm. Fixed yeast cells are attached to coverslips and hybridized with a mixture of FISH probes, each conjugated to several fluorescent dyes. Images of cells are acquired in 3D and maximally projected for single-molecule analysis. Diffraction-limited labeled mRNAs are observed as bright fluorescent spots and can be quantified using a spot-detection algorithm. FISH preserves the spatial distribution of cellular RNA distribution within the cell and the stochastic fluctuations in individual cells that can lead to phenotypic differences within a clonal population. This information, however, is lost if the RNA content is measured on a population of cells by using reverse transcriptase PCR, microarrays or high-throughput sequencing. The FISH procedure and image acquisition described here can be completed in 3 d.

Trcek, Tatjana; Chao, Jeffrey A; Larson, Daniel R; Park, Hye Yoon; Zenklusen, Daniel; Shenoy, Shailesh M; Singer, Robert H

2014-01-01

64

Use of Fluorescence In situ Hybridization to Detect and Monitor Transfected and Amplified Sequences in Recombinant CHO Cells.  

National Technical Information Service (NTIS)

Fluorescence In Situ Hybridization (FISH) can be used routinely to detect and monitor foreign DNA sequences in recombinant mammalian cells. Non-radioactive hybridization and detection of foreign DNA can be performed within 1-2 days, in contrast to weeks o...

M. G. Pallavicini P. S. DeTeresa F. M. Wurm

1988-01-01

65

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

Microsoft Academic Search

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

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

2008-01-01

66

Fluorescence In Situ Hybridization-Based Karyotyping of Soybean Translocation Lines  

PubMed Central

Soybean (Glycine max [L.] Merr.) is a major crop species and, therefore, a major target of genomic and genetic research. However, in contrast to other plant species, relatively few chromosomal aberrations have been identified and characterized in soybean. This is due in part to the difficulty of cytogenetic analysis of its small, morphologically homogeneous chromosomes. The recent development of a fluorescence in situ hybridization –based karyotyping system for soybean has enabled our characterization of most of the chromosomal translocation lines identified to date. Utilizing genetic data from existing translocation studies in soybean, we identified the chromosomes and approximate breakpoints involved in five translocation lines.

Findley, Seth D.; Pappas, Allison L.; Cui, Yaya; Birchler, James A.; Palmer, Reid G.; Stacey, Gary

2011-01-01

67

Characterization of supernumerary ring marker chromosomes by fluorescence in situ hybridization (FISH)  

SciTech Connect

Five cases with small supernumerary ring chromosomes are characterized at the molecular level. Routine chromosome banding analysis was insufficient for identification of the ring chromosomes, and none of them was DA/DAPI positive. Fluorescence in situ hybridization utilizing repetitive centromeric probes for all chromosomes has determined that one of these five ring chromosomes originates in each of chromosomes 4, 7, 8, 9, and 20. Chromosome painting with chromosome-specific libraries has confirmed this and excluded the involvement of additional chromosomes in the rearrangements. 30 refs., 3 figs., 2 tabs.

Blennow, E.; Nordenskjoeld, M. (Karolinska Hospital (Sweden)); Asadi, E. (Region Hospital, Oerebro (Sweden)); Anneren, G.; Berggren, E.; Nordenskjoeld, M.

1993-08-01

68

Quantitative fluorescence in situ hybridization of Aureobasidium pullulans on microscope slides and leaf surfaces.  

PubMed Central

A 21-mer oligonucleotide probe designated Ap665, directed at the 18S rRNA of Aureobasidium pullulans and labelled with five molecules of fluorescein isothiocyanate, was applied by fluorescence in situ hybridization (FISH) to populations of the fungus on slides and apple leaves from growth chamber seedlings and orchard trees. In specificity tests that included Ap665 and a similarly labelled universal probe and the respective complementary probes as controls, the hybridization signal was strong for Ap665 reactions with 12 A. pullulans strains but at or below background level for 98 other fungi including 82 phylloplane isolates. Scanning confocal laser microscopy was used to confirm that the fluorescence originated from the cytoplasmic matrix and to overcome limitations imposed on conventional microscopy by leaf topography. Images were recorded with a cooled charge-coupled device video camera and digitized for storage and manipulation. Image analysis was used to verify semiquantitative fluorescence ratings and to demonstrate how the distribution of the fluorescence signal in specific interactions (e.g., Ap665 with A. pullulans cells) could be separated at a given probability level from nonspecific fluorescence (e.g., in interactions of Ap665 with Cryptococcus laurentii cells) of an overlapping population. Image analysis methods were used also to quantify epiphytic A. pullulans populations based on cell number or percent coverage of the leaf surface. Under some conditions, leaf autofluorescence and the release of fluorescent compounds by leaves during the processing for hybridization decreased the signal-to-noise ratio. These effects were reduced by the use of appropriate excitation filter sets and fixation conditions. We conclude that FISH can be used to detect and quantify A. pullulans cells in the phyllosphere.

Li, S; Spear, R N; Andrews, J H

1997-01-01

69

Development of single-cell array for large-scale DNA fluorescence in situ hybridization  

PubMed Central

DNA fluorescence in situ hybridization (FISH) is a powerful cytogenetic assay, but conventional sample-preparation methods for FISH do not support large-scale high-throughput data acquisition and analysis, which are potentially useful for several biomedical applications. To address this limitation, we have developed a novel FISH sample-preparation method based on generating a centimetre-sized cell array, in which all cells are precisely positioned and separated from their neighbours. This method is simple and easy and capable of patterning nonadherent human cells. We have successfully performed DNA FISH on the single-cell arrays, which facilitate analysis of FISH results with the FISH-FINDER computer program.

Liu, Yingru; Kirkland, Brett; Shirley, James; Wang, Zhibin; Zhang, Peipei; Stembridge, Jacquelyn; Wong, Wilson; Takebayashi, Shin-ichiro; Gilbert, David M.; Lenhert, Steven

2013-01-01

70

Improved detection of soil microorganisms using fluorescence in situ hybridization (FISH) and catalyzed reporter deposition (CARD-FISH)  

Microsoft Academic Search

In recent years, methods of molecular microbiology have been used for the investigation of soil microbial diversity. Fluorescence in situ hybridization (FISH) represents a method which allows a specific staining and enumeration of soil microorganisms by using fluorescent-labelled oligonucleotide probes. However, the detection of FISH-stained cells is often affected by strong autofluorescence of the background, especially in samples of the

Thilo Eickhorst; Rolf Tippkötter

2008-01-01

71

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

PubMed

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

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

2011-06-01

72

Simultaneous specific in planta visualization of root-colonizing fungi using fluorescence in situ hybridization (FISH).  

PubMed

In planta detection of mutualistic, endophytic, and pathogenic fungi commonly colonizing roots and other plant organs is not a routine task. We aimed to use fluorescence in situ hybridization (FISH) for simultaneous specific detection of different fungi colonizing the same tissue. We have adapted ribosomal RNA (rRNA) FISH for visualization of common mycorrhizal (arbuscular- and ectomycorrhiza) and endophytic fungi within roots of different plant species. Beside general probes, we designed and used specific ones hybridizing to the large subunit of rRNA with fluorescent dyes chosen to avoid or reduce the interference with the autofluorescence of plant tissues. We report here an optimized efficient protocol of rRNA FISH and the use of both epifluorescence and confocal laser scanning microscopy for simultaneous specific differential detection of those fungi colonizing the same root. The method could be applied for the characterization of other plant-fungal interactions, too. In planta FISH with specific probes labeled with appropriate fluorescent dyes could be used not only in basic research but to detect plant colonizing pathogenic fungi in their latent life-period. PMID:24221902

Vági, Pál; Knapp, Dániel G; Kósa, Annamária; Seress, Diána; Horváth, Aron N; Kovács, Gábor M

2014-05-01

73

Fluorescence in situ hybridization evaluation of chromosome deletion patterns in prostate cancer.  

PubMed Central

Various nonrandom chromosomal aberrations have been identified in prostate carcinoma. These aberrations include deletions of several chromosome regions, particularly the chromosome 8 short arm. Large-scale numerical aberrations, reflected in aberrant DNA ploidy, are also found in a minority of cases. However, it is unclear whether prostate carcinomas contain aberrations of certain chromosome regions that are deleted frequently in other common types of cancer. In this study, we performed dual-color fluorescence in situ hybridization on intact nuclei from touch preparations of 16 prostate cancers. Chromosome copy number was determined using pericentromeric probes, whereas potential chromosome arm deletions were evaluated using yeast artificial chromosome (YAC) and P1 probes. Two YAC probes targeted chromosome 8 short arm regions known to be deleted frequently in prostate cancer. Other YACs and P1s were for chromosome regions, including 1p22, 3p14, 6q21, 9p21, and 22q12, that are deletion targets in a variety of cancers although not extensively studied in prostate cancer. Hybridization efficiencies and signal intensities were excellent for both repeat sequence (alpha-satellite) and single, copy (YAC and P1) fluorescence in situ hybridization probes. Of 16 prostate cancers, 11 had clonal aberrations of 1 or more of the 13 chromosome regions evaluated, and 10 cases (62.5%) had 8p deletions, including 4 cases with 8p deletion in virtually all cells and aneuploidy in only a subset of those deleted cells. Deletions at 3p14, 6q21, and 22q12 were identified in 2, 1, and 1 case, respectively, and each of those cases had a similarly sized cell population with 8p deletion. These studies confirm 8p deletion in the majority of prostate carcinomas. 8p deletions appear to be early events in prostate tumorigenesis, often antedating aneuploidy. Fluorescence in situ hybridization strategies incorporating pericentromeric and single-copy regional chromosome probes offer a powerful and efficient means for determining frequency and progression of oncogenetic events in prostate cancer. Images Figure 1

Huang, S. F.; Xiao, S.; Renshaw, A. A.; Loughlin, K. R.; Hudson, T. J.; Fletcher, J. A.

1996-01-01

74

Combined fluorescence in situ hybridization and PRINS for the analysis of the Dystrophin gene.  

PubMed

Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy are caused in most cases by deletions of the DMD gene. These rearrangements are detectable in affected boys and men by a simple multiplex polymerase chain reaction approach. However, this technique is not able to disclose DMD deletions in heterozygous female carriers, and different approaches must be used in these cases. Here, we describe an approach based on the combined use of primed in situ labeling and fluorescence in situ hybridization techniques for the detection of single DMD exons in fixed metaphase chromosomes and interphase nuclei of both male and female subjects, suggesting the usefulness of this tool in the detection of small intragenic deletions of the DMD gene. PMID:16861757

Stuppia, Liborio; La Sala, Dario; Cinti, Caterina

2006-01-01

75

Analysis of HER2 Gene Amplification Using an Automated Fluorescence in Situ Hybridization Signal Enumeration System  

PubMed Central

The HER2 gene, amplified in 10 to 35% of invasive human breast carcinomas, has prognostic and therapeutic implications. Fluorescent in situ hybridization is one method currently used for assessing HER2 status, but fluorescent in situ hybridization involves the time-consuming step of manual signal enumeration. To address this issue, Vysis has developed an automated signal enumeration system, Vysis AutoVysion. A multicenter, blinded study was conducted on 39 formalin-fixed, paraffin-embedded invasive breast carcinoma specimens, including 20 HER2 nonamplified and 19 HER2 amplified (weakly to highly amplified), provided in duplicate to each study site for analysis. Calculation of the HER2/CEP17 ratio and the hands-on time of both manual and automated enumeration approaches were compared. Overall agreement of HER2 classification results (positive and negative) was 92.5% (196 of 212). The Vysis AutoVysion System requires manual enumeration for cases with scanner results within the ratio range of 1.5 to 3.0. When the data in this range are excluded, the agreement between manual and scanner results is 98.8% (169 of 171). The average Vysis AutoVysion System hands-on time per slide was 4.59 versus 7.47 minutes for manual signal enumeration (savings of 2.88 minutes/slide). These data suggest that the Vysis AutoVysion System can correctly classify specimens and may increase the overall efficiency of HER2 testing.

Stevens, Rachel; Almanaseer, Imad; Gonzalez, Miguel; Caglar, Derin; Knudson, Ryan A.; Ketterling, Rhett P.; Schrock, Daniel S.; Seemayer, Thomas A.; Bridge, Julia A.

2007-01-01

76

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

SciTech Connect

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.

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

77

Assignment of 55 novel cosmids to seven subregions of chromosome 13 using fluorescence in situ hybridization  

SciTech Connect

Fifty-five individual cosmids isolated from a chromosome 13-specific library have been regionally assigned using fluorescence in situ hybridization. These cosmids represent novel DNA markers that can be added to the limited number of DNA probes already available for this chromosome. Individual cosmids mapped along the length of the chromosome with almost equal distribution except that there appears to be an excess of probes from the distal 13q33-q34 region. Only two cosmids did not map back to chromosome 13, indicating that at least 96% of cosmids in this library are from this chromosome. The library is generally available as gridded colonies on nylon membranes suitable for hybridization screening. 12 refs., 2 figs.

Hawthorn, L.; Cowell, J.K. (Institute of Child Health, London (United Kingdom)); Nizetic, D.; Lehrach, H. (Genome Analysis Lab., London (United Kingdom))

1994-05-01

78

Recent advances in rice genome and chromosome structure research by fluorescence in situ hybridization (FISH)  

PubMed Central

Fluorescence in situ hybridization (FISH) is an effective method for the physical mapping of genes and repetitive DNA sequences on chromosomes. Physical mapping of unique nucleotide sequences on specific rice chromosome regions was performed using a combination of chromosome identification and highly sensitive FISH. Increases in the detection sensitivity of smaller DNA sequences and improvements in spatial resolution have ushered in a new phase in FISH technology. Thus, it is now possible to perform in situ hybridization on somatic chromosomes, pachytene chromosomes, and even on extended DNA fibers (EDFs). Pachytene-FISH allows the integration of genetic linkage maps and quantitative chromosome maps. Visualization methods using FISH can reveal the spatial organization of the centromere, heterochromatin/euchromatin, and the terminal structures of rice chromosomes. Furthermore, EDF-FISH and the DNA combing technique can resolve a spatial distance of 1 kb between adjacent DNA sequences, and the detection of even a 300-bp target is now feasible. The copy numbers of various repetitive sequences and the sizes of various DNA molecules were quantitatively measured using the molecular combing technique. This review describes the significance of these advances in molecular cytology in rice and discusses future applications in plant studies using visualization techniques.

Ohmido, Nobuko; Fukui, Kiichi; Kinoshita, Toshiro

2010-01-01

79

Visualizing the Needle in the Haystack: In Situ Hybridization With Fluorescent Dendrimers  

PubMed Central

In situ hybridization with 3DNA™ dendrimers is a novel tool for detecting low levels of mRNA in tissue sections and whole embryos. Fluorescently labeled dendrimers were used to identify cells that express mRNA for the skeletal muscle transcription factor MyoD in the early chick embryo. A small population of MyoD mRNA positive cells was found in the epiblast prior to the initiation of gastrulation, two days earlier than previously detected using enzymatic or radiolabeled probes for mRNA. When isolated from the epiblast and placed in culture, the MyoD mRNA positive cells were able to differentiate into skeletal muscle cells. These results demonstrate that DNA dendrimers are sensitive and precise tools for identifying low levels of mRNA in single cells and tissues.

Baytion, Michael; Perlman, Jordanna; Neely, Christine; Hearon, Bridget; Nilsen, Thor; Getts, Robert; Kadushin, James; George-Weinstein, Mindy

2004-01-01

80

Fluorescence in situ hybridization techniques for the rapid detection of genetic prognostic factors in neuroblastoma  

PubMed Central

Neuroblastoma is the commonest extracranial solid tumour in children. There are a number of molecular genetic features known which are of prognostic importance and which are used to direct therapy. Identification and targeting of high-risk individuals with intensive therapeutic regimens may allow an improvement in survival rates. The most powerful biological parameters associated with prognosis in this malignancy are chromosomal changes, especially MYCN amplification, deletion of chromosome 1p and aneuploidy. Rapid characterization of these aberrations at the time of diagnosis is paramount if stratification according to risk group is to be achieved. This paper describes the rapid detection of del(1p), MYCN amplification and trisomy using interphase fluorescence in situ hybridization on imprints from fresh tumour biopsies. The results are related to those obtained by standard molecular methods and karyotyping. © 2000 Cancer Research Campaign

Taylor, C P F; Bown, N P; McGuckin, A G; Lunec, J; Malcolm, A J; Pearson, A D J; Sheer, D

2000-01-01

81

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

PubMed Central

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.

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

2013-01-01

82

Substrate uptake in extremely halophilic microbial communities revealed by microautoradiography and fluorescence in situ hybridization.  

PubMed

The combination of fluorescence in situ hybridization and microautoradiography (FISH-MAR approach) was applied to brine samples of a solar saltern crystallizer pond from Mallorca (Spain) where the simultaneous occurrence of Salinibacter spp. and the conspicuous square Archaea had been detected. Radioactively labeled bicarbonate, acetate, glycerol, and an amino acid mixture were tested as substrates for the microbial populations inhabiting such brines. The results indicated that hitherto uncultured 'square Archaea' do actively incorporate amino acids and acetate. However, Salinibacter spp. only showed amino acid incorporation in pure culture, but no evidence of such activity in their natural environment could be demonstrated. No glycerol incorporation was observed for any component of the microbial community. PMID:12820037

Rosselló-Mora, Ramon; Lee, Natuschka; Antón, Josefa; Wagner, Michael

2003-10-01

83

Detection of genetic alterations in micrometastatic cells in bone marrow of cancer patients by fluorescence in situ hybridization  

Microsoft Academic Search

Detection of micrometastatic tumor cells in bone marrow of cancer patients has been shown to be of prognostic significance. To further characterize these cells, we combined antibody labeling and fluorescence in situ hybridization (FISH). For detection of numerical changes of chromosome 17, nine patients with proven breast cancer whose bone marrow contained epithelial tumor cells were evaluated. Epithelial cells were

Peter Müller; Dorothea Weckermann; Gert Riethmüller; Günter Schlimok

1996-01-01

84

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

PubMed Central

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.

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

2011-01-01

85

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2013 CFR

...AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Immunology Laboratory Equipment and Reagents § 866.4700 Automated fluorescence in situ...

2013-04-01

86

Cytogenetic and fluorescence in situ hybridization studies in 60 patients with multiple myeloma and plasma cell leukemia  

Microsoft Academic Search

We report cytogenetic results in a series of 60 patients affected with multiple myeloma (MM) and plasma cell leukemia (PCL) and compare the results with those previously reported. In our series, a total of 41% of MM patients and 71% of PCL patients displayed chromosome abnormalities. To evaluate the clinical value of monosomy 18, we obtained fluorescence in situ hybridization

Elisabet Lloveras; Isabel Granada; Lurdes Zamora; Blanca Espinet; Lourdes Florensa; Carles Besses; Marisol Xandri; Maria Encarnación Pérez-Vila; Fuensanta Millŕ; Soledad Woessner; Francesc Solé

2004-01-01

87

Detection of micro-organisms in soil after in situ hybridization with rRNA-targeted, fluorescently labelled oligonucleotides  

Microsoft Academic Search

rRNA sequences were used as targets for synthetic oligonucleotides labelled with the fluorescent dye tetramethylrhodamine isothiocyanate (Tritc) for in situ hybridizations to detect micro-organisms directly in soils that have different contents of soil minerals and organic material. Introduced Pseudomonas aeruginosa cells were directly fixed in soils and applied to slides after separation of large soil minerals only. Remaining soil minerals

DITTMAR HAHN; RUDOLF I. AMANN; WOLFGANG LUDWIG; ANTOON D. L. AKKERMANS; KARL-HEINZ SCHLEIFER

1992-01-01

88

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

SciTech Connect

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.

Lucas, J.N.; Straume, T.

1995-10-01

89

Identification of duchenne muscular dystrophy female carriers by fluorescence in situ hybridization and RT-PCR.  

PubMed

Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder caused by mutations in the dystrophin DMD gene located at Xp21.1 region. Up to 65% of the patients present dystrophin gene deletions. Mothers of DMD patients have a two-thirds chance of carrying a dystrophin mutation. The female carrier will transmit the disease gene to half of her sons and half of her daughters. As the recurrence risk for the disease is extremely high, it is very important to detect carrier status among female relatives of the patients to bring an adequate genetic counseling. In this work, results from two methods to identify female carriers are presented. One method is a multicolor fluorescence in situ hybridization (FISH) assay, and the other is reverse transcriptase-polymerase chain reaction (RT-PCR). We showed that FISH is an efficient, sensitive method that brings confident results to detect DMD female carriers as compared to RT-PCR. PMID:18471087

Velázquez-Wong, Ana Claudia; Hernández-Huerta, César; Márquez-Calixto, Areli; Hernández-Aguilar, Fidel Omar; Rodríguez-Cruz, Maricela; Salamanca-Gómez, Fabio; Coral-Vázquez, Ramón

2008-06-01

90

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

NASA Astrophysics Data System (ADS)

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.

Yi, Dingrong; Xie, Shaochuan

2013-12-01

91

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

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.

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

1999-01-01

92

Evaluation of the Phylogenetic Diversity of Prokaryotic Microorganisms in Sphagnum Peat Bogs by Means of Fluorescence In Situ Hybridization (FISH)  

Microsoft Academic Search

The microbial population of sphagnum peat bogs of northern Russia was analyzed with respect to the presence and cell numbers\\u000a of representatives of particular phylogenetic groups of prokaryotes by means of in situ hybridization with fluorescently labeled\\u000a group-specific rRNA-targeted oligonucleotide probes with broad detection spectra. The total number of cells that hybridized\\u000a with universal Archaea- and Bacteria-specific probes varied, in

T. A. Pankratov; S. E. Belova; S. N. Dedysh

2005-01-01

93

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

PubMed Central

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 probes that allowed identification of approximately 95% of those pathogens typically associated with bacteremia were produced. The sensitivity and specificity of these probes were 100%. From all 115 blood cultures, microorganisms were grown after 1 day and identification to the family, genus, or species level was achieved after 1 to 3 days while 111 samples (96.5%) were similarly identified by FISH within 2.5 h. Staphylococci were identified in 62 of 62 samples, streptococci and enterococci were identified in 19 of 20 samples, gram-negative rods were identified in 28 of 30 samples, and fungi were identified in two of two samples. Thus, FISH is an appropriate method for identification of pathogens grown in blood cultures from septicemic patients.

Kempf, Volkhard A. J.; Trebesius, Karlheinz; Autenrieth, Ingo B.

2000-01-01

94

Determination of the ruminant origin of bone particles using fluorescence in situ hybridization (FISH)  

PubMed Central

Molecular biology techniques such as PCR constitute powerful tools for the determination of the taxonomic origin of bones. DNA degradation and contamination by exogenous DNA, however, jeopardise bone identification. Despite the vast array of techniques used to decontaminate bone fragments, the isolation and determination of bone DNA content are still problematic. Within the framework of the eradication of transmissible spongiform encephalopathies (including BSE, commonly known as “mad cow disease”), a fluorescence in situ hybridization (FISH) protocol was developed. Results from the described study showed that this method can be applied directly to bones without a demineralisation step and that it allows the identification of bovine and ruminant bones even after severe processing. The results also showed that the method is independent of exogenous contamination and that it is therefore entirely appropriate for this application.

Lecrenier, M. C.; Ledoux, Q.; Berben, G.; Fumiere, O.; Saegerman, C.; Baeten, V.; Veys, P.

2014-01-01

95

A physical map of chromosome 20 established using fluorescence in situ hybridization and digital image analysis  

SciTech Connect

The physical locations of 46 cosmid clones and 21 P1 clones were determined along the chromosome 20 axis relative to the p terminus (FLpter) using fluorescence in situ hybridization (FISH) and digital image microscopy. The cosmid clones were selected from the chromosomally enriched library LA20NC01. Nine P1 clones were selected from a pooled DuPont genomic library using PCR with primer pairs selected to amplify genetically mapped sequence-tagged sites. This information was used to relate the physical map to the genetic map. Twelve P1 clones were selected from the same library using PCR primer pairs that amplified known genes. Two of these, E2F and BCLX, had not been mapped previously. 14 refs., 1 fig., 2 tabs.

Stokke, T.; Collins, C.; Kuo, W.L. [Univ. of California, San Francisco, CA (United States)] [and others] [Univ. of California, San Francisco, CA (United States); and others

1995-03-01

96

Detection of aneuploidy in human spermatozoa of normal semen donors by fluorescence in situ hybridization.  

PubMed Central

We have studied human spermatozoa from 24 normal, healthy unexposed men, 18 of whom were semen donors at the Sperm Bank in Turku, using multicolor fluorescence in situ hybridization with two chromosome-specific probes. The possible age-related increase in aneuploidy frequencies was assessed. Ten thousand spermatozoa were scored per individual for the presence of hyperploid, i.e., disomic and diploid, cells. The overall hybridization efficiency was 98.8%. The frequency of spermatozoa with two chromosome 1 signals was 11.5 +/- 5.2/10,000. The frequency of spermatozoa with two chromosome 7 signals was 6.4 +/- 3.9/10,000. Diploidy was present in 15.0 +/- 8.9/10,000 spermatozoa. Interindividual variation was quite large. No statistically significant correlation between age of the donors (range = 20-46 years) and the frequency of hyperploid spermatozoa was observed. The results give background information on the incidence of hyperploid spermatozoa in unexposed men and encourage the use of this novel technique of future studies on genetic effects in men exposed to potentially aneuploidogenic agents.

Lahdetie, J; Ajosenpaa-Saari, M; Mykkanen, J

1996-01-01

97

Fluorescent in situ hybridization (FISH) assessment of chromosome copy number in sperm  

SciTech Connect

Approximately 15% of all recognized pregnancies end in spontaneous abortions. The overall frequency of chromosome abnormalities in spontaneous abortions is approximately 50%. Thus aneuploidy is a significant cause of fetal wastage. In addition, structural and numerical abnormalities of chromosomes can also lead to birth defects, developmental delay, mental retardation and infertility. Conventional cytogenetic analysis via GTG- and other banding techniques is a powerful tool in the elucidation of the nature of chromosomal abnormalities. Fluorescent in situ hybridization (FISH) enables detection of numerical chromosomal abnormalities, especially trisomies, in intact cells. Using FISH and commercially available biotin-labeled probes, we have initiated a prospective study to assess specific chromosome copy number of preparations of unstained smears from men referred for a male infertility evaluation as well as smears from normal control males chosen randomly from the sample of sperm donors. A total of approximately 19,000 sperm nuclei have been examined thus far. Of those suitable for analysis, 7382 (38.75%) were normal possessing one copy of chromosome 8, 155 (0.81%) were disomic, and 15 (0.079%) had more than two copies of chromosome 8. Comparisons with data available in the literature will be discussed. Work is ongoing to increase the efficiency of hybridization using both reported and previously untried pretreatment and fixation protocols. We have also initiated studies using multicolor FISH with various chromosome enumeration probes. The assay described here is a potentially powerful tool for detecting rare events such as spontaneous germ cell aneuploidy, aneuploidy detected in semen from men with carcinoma in situ of the testis and aneuploidy induced by potential environmental genotoxicants. It can also be utilized for segregation analysis and for correlating chromosome copy number with germ cell morphology.

Sheu, M.; Sigman, M.; Mark, H.F.L. [Brown Univ. School of Medicine, Providence, RI (United States)

1994-09-01

98

Analysis of Sex-mismatched Human Corneal Transplants by Fluorescence in situ Hybridization of the Sex-chromosomes  

Microsoft Academic Search

The fate of the cells of corneal transplants has been controversial from the early days of keratoplasty. Various methods such as histological evaluation, radiolabeling of donor cells or Barr-body analysis have been applied to clarify the issue. However, the question whether the transplanted cells are replaced or survive, remains unsolved.In this study, we applied fluorescence in situ hybridization (FISH) of

GREGOR WOLLENSAK; WILLIAM RICHARD GREEN

1999-01-01

99

FISHIS: Fluorescence In Situ Hybridization in Suspension and Chromosome Flow Sorting Made Easy  

PubMed Central

The large size and complex polyploid nature of many genomes has often hampered genomics development, as is the case for several plants of high agronomic value. Isolating single chromosomes or chromosome arms via flow sorting offers a clue to resolve such complexity by focusing sequencing to a discrete and self-consistent part of the whole genome. The occurrence of sufficient differences in the size and or base-pair composition of the individual chromosomes, which is uncommon in plants, is critical for the success of flow sorting. We overcome this limitation by developing a robust method for labeling isolated chromosomes, named Fluorescent In situ Hybridization In suspension (FISHIS). FISHIS employs fluorescently labeled synthetic repetitive DNA probes, which are hybridized, in a wash-less procedure, to chromosomes in suspension following DNA alkaline denaturation. All typical A, B and D genomes of wheat, as well as individual chromosomes from pasta (T. durum L.) and bread (T. aestivum L.) wheat, were flow-sorted, after FISHIS, at high purity. For the first time in eukaryotes, each individual chromosome of a diploid organism, Dasypyrum villosum (L.) Candargy, was flow-sorted regardless of its size or base-pair related content. FISHIS-based chromosome sorting is a powerful and innovative flow cytogenetic tool which can develop new genomic resources from each plant species, where microsatellite DNA probes are available and high quality chromosome suspensions could be produced. The joining of FISHIS labeling and flow sorting with the Next Generation Sequencing methodology will enforce genomics for more species, and by this mightier chromosome approach it will be possible to increase our knowledge about structure, evolution and function of plant genome to be used for crop improvement. It is also anticipated that this technique could contribute to analyze and sort animal chromosomes with peculiar cytogenetic abnormalities, such as copy number variations or cytogenetic aberrations.

Giorgi, Debora; Farina, Anna; Grosso, Valentina; Gennaro, Andrea; Ceoloni, Carla; Lucretti, Sergio

2013-01-01

100

Investigation of chicken intestinal bacterial communities by 16S rRNA targeted fluorescence in situ hybridization  

Microsoft Academic Search

The aim of the investigation was to quantify selected dominant bacterial groups in the chicken intestinal tract. Conventional\\u000a production was used as model and the effect of the supplement with Salinomycin was evaluated. Hybridization conditions were\\u000a optimized for published probes with respect to a panel of reference bacteria. In chicken intestinal samples bacteria were\\u000a quantified by fluorescence in situ hybridization

K. N. Olsen; M. Henriksen; M. Bisgaard; O. L. Nielsen; H. Christensen

2008-01-01

101

DNA Mimics for the Rapid Identification of Microorganisms by Fluorescence in situ Hybridization (FISH)  

PubMed Central

Fluorescence in situ hybridization (FISH) is a well-established technique that is used for a variety of purposes, ranging from pathogen detection in clinical diagnostics to the determination of chromosomal stability in stem cell research. The key step of FISH involves the detection of a nucleic acid region and as such, DNA molecules have typically been used to probe for the sequences of interest. However, since the turn of the century, an increasing number of laboratories have started to move on to the more robust DNA mimics methods, most notably peptide and locked nucleic acids (PNA and LNA). In this review, we will cover the state-of-the-art of the different DNA mimics in regard to their application as efficient markers for the presence of individual microbial cells, and consider their potential advantages and pitfalls. Available PNA probes are then reassessed in terms of sensitivity and specificity using rRNA databases. In addition, we also attempt to predict the applicability of DNA mimics in well-known techniques attempting to detect in situ low number of copies of specific nucleic acid sequences such as catalyzed reporter deposition (CARD) and recognition of individual genes (RING) FISH.

Cerqueira, Laura; Azevedo, Nuno F.; Almeida, Carina; Jardim, Tatiana; Keevil, Charles William; Vieira, Maria J.

2008-01-01

102

Fluorescent in situ hybridization of synaptic proteins imaged with super-resolution STED microscopy.  

PubMed

Super-resolution fluorescence microscopy is still a developing field. One of the limitations has been that standard labeling assays, which had been developed for conventional imaging, must be adjusted and optimized for each super-resolution method. These methods are more sensitive to noise, and require more intense labeling than conventional microscopy, which is not always trivial to achieve. Here, we describe the use of stimulation-emission depletion (STED) microscopy to locate messenger RNAs (mRNAs) in single neurons with high spatial precision. We address several technical difficulties we encountered in using fluorescent in situ hybridization (FISH) for STED imaging. We optimized the experimental protocol to detect mRNAs and proteins simultaneously, by performing FISH and immunostaining on the same samples. We tested our imaging approach in primary hippocampal neurons, studying the mRNAs of three important presynaptic proteins (synaptobrevin, synaptotagmin, and synaptophysin). Our approach allowed us to relate changes in mRNA levels and localization to neuronal physiology, under different activity regimes and also during neuronal development. We conclude that FISH can be performed efficiently using super-resolution techniques. This should contribute significantly to the clarification of the molecular mechanisms that govern mRNA distribution and dynamics within cells. Microsc. Res. Tech. 77:517-527, 2014. © 2014 Wiley Periodicals, Inc. PMID:24723361

Zhang, William I; Röhse, Heiko; Rizzoli, Silvio O; Opazo, Felipe

2014-07-01

103

Metaphase and interphase fluorescence in situ hybridization mapping of the rice genome with bacterial artificial chromosomes.  

PubMed Central

Fluorescence in situ hybridization (FISH) is a powerful tool for physical mapping in human and other mammalian species. However, application of the FISH technique has been limited in plant species, especially for mapping single- or low-copy DNA sequences, due to inconsistent signal production in plant chromosome preparations. Here we demonstrate that bacterial artificial chromosome (BAC) clones can be mapped readily on rice (Oryza sativa L.) chromosomes by FISH. Repetitive DNA sequences in BAC clones can be suppressed efficiently by using rice genomic DNA as a competitor in the hybridization mixture. BAC clones as small as 40 kb were successfully mapped. To demonstrate the application of the FISH technique in physical mapping of plant genomes, both anonymous BAC clones and clones closely linked to a rice bacterial blight-resistance locus, Xa21, were chosen for analysis. The physical location of Xa21 and the relationships among the linked clones were established, thus demonstrating the utility of FISH in plant genome analysis. Images Fig. 1

Jiang, J; Gill, B S; Wang, G L; Ronald, P C; Ward, D C

1995-01-01

104

Method for multiplex cellular detection of mRNAs using quantum dot fluorescent in situ hybridization  

PubMed Central

The photostability and narrow emission spectra of non-organic quantum dot fluorophores (QDs) make them desirable candidates for fluorescent in situ hybridization (FISH) to study the expression of specific mRNA transcripts. We developed a novel method for direct QD labeling of modified oligonucleotide probes through streptavidin and biotin interactions, as well as protocols for their use in multiple-label FISH. We validated this technique in mouse brainstem sections. The subcellular localization of the vesicular monoamine transporter (Vmat2) mRNA corresponds when using probes labeled with two different QDs in the same hybridization. We developed protocols for combined direct QD FISH and QD immunohistochemical labeling within the same neurons as well as for simultaneous study of the subcellular distribution of multiple mRNA targets. We demonstrated increased sensitivity of FISH using QDs in comparison with organic fluorophores. These techniques gave excellent histological results both for multiplex FISH and combined FISH and immunohistochemistry. This approach can facilitate the ultrasensitive simultaneous study of multiple mRNA and protein markers in tissue culture and histological section.

Chan, PokMan; Yuen, Tony; Ruf, Frederique; Gonzalez-Maeso, Javier; Sealfon, Stuart C.

2005-01-01

105

Utility of fluorescence in situ hybridization for ploidy and p57 immunostaining in discriminating hydatidiform moles.  

PubMed

Discrimination between complete moles (CMs), partial moles (PMs), and hydropic abortions (HAs) is important as the risk of persistent gestational trophoblastic disease (GTD) differs for each condition. We evaluated whether ancillary fluorescence in situ hybridization (FISH) with a set of chromosome enumeration probes (CEP) for chromosomes X, Y, and 17 and p57 immunostaining could improve the clinical diagnosis. Forty-one products of conception (POC) were reclassified according to clinical performance, morphology, p57 immunostaining results, and FISH results. The accuracy of histological examination alone was 85% for the original diagnosis. FISH analysis showed diploidy in 19 of 20 CMs and triploidy in 4 of 6 PMs. The concordance rate was 92.5% on using the CEP probes. p57 Staining was negative in all CMs and positive in all PMs and HAs. Chromosomal abnormality was detected in 3 cases of HA by using FISH. In conclusion, combined p57 immunostaining and FISH with a set of 3 CEP probes for chromosomes X, Y, and 17 could be useful in the classification of hydatidiform moles. PMID:24613849

Chen, Kuang-Hua; Hsu, Sheng-Chi; Chen, Hou-Yu; Ng, Kwai-Fong; Chen, Tse-Ching

2014-04-01

106

Correlations between arsenic in Maine groundwater and microbial populations as determined by fluorescence in situ hybridization.  

PubMed

Arsenic is known to cause serious health effects when consumed in drinking water. In the state of Maine, approximately half of the population relies on private groundwater wells for their drinking water. Of those wells, as many as 13% may contain arsenic levels above the current EPA maximum contaminant level of 10 microgl(-1). Microorganisms can potentially contribute to arsenic release into groundwater through several mechanisms. Some can reduce arsenate to arsenite, which is more toxic and may be more mobile. Sulfurospirillum species NP4, which was isolated from well water, respires arsenate and could act in this way. Microorganisms can also act indirectly by reducing bedrock surface coatings, such as iron oxyhydroxides, that adsorb arsenic in the groundwater environment. The genus Geobacter contains many species that are capable of iron reduction that could play a role in the indirect release of arsenic into groundwater. Water samples from Northport, ME and the Branch Lake region of Ellsworth, ME, which both have elevated groundwater arsenic levels, have been probed using fluorescence in situ hybridization (FISH), to determine the percentage of the population that is NP4 and the percentage that are Geobacter species. Geobacter abundance correlates well with the total arsenic concentration indicating that indirect mechanisms could be important in releasing arsenic. NP4 appears to be reducing arsenate since its prevalence correlates well with arsenite, the end product of arsenate respiration. PMID:16310822

Weldon, Jennifer M; MacRae, Jean D

2006-04-01

107

Fluorescence in situ Hybridization Analysis of Circulating Tumor Cells in Metastatic Prostate Cancer  

PubMed Central

Purpose To assess the feasibility of characterizing gene copy number alteration by fluorescence in situ hybridization of circulating tumor cells (CTC) isolated using the CellSearch system in patients with progressive castration resistant metastatic prostate cancer (CRPC). Experimental Design We used probe combinations that included the androgen receptor (AR) and MYC genes for FISH analysis of CTC samples collected from 77 men with metastatic CRPC. Results High-level chromosomal amplification of AR was detected in 37.5% of samples analyzed, and relative gain of MYC in 55.8%. No such abnormalities were detected in samples with CTC counts of less than 10, reflecting ascertainment difficulty in these lower count samples. Conclusion The CTC isolated from our patient cohort present a very similar molecular cytogenetic profile to that reported for late-stage tumors, and thus demonstrate that analysis of CTC can be a valuable, noninvasive surrogate for routine tumor profiling. Furthermore, we demonstrate that as many as 50% of these patients have substantial amplification of the AR locus, indicating that androgen signaling continues to play an important role in late-stage prostate cancer.

Leversha, Margaret A.; Han, Jialian; Asgari, Zahra; Danila, Daniel C.; Lin, Oscar; Gonzalez-Espinoza, Rita; Anand, Aseem; Lilja, Hans; Heller, Glenn; Fleisher, Martin; Scher, Howard I.

2010-01-01

108

Identification of pathogens in mastitis milk samples with fluorescent in situ hybridization.  

PubMed

Traditionally, the bacteriological examination of mastitis milk samples is performed by culture followed by biochemical tests on the cultured bacteria to allow identification of the causative pathogen. Depending on the species involved, this classic identification is time-consuming compared to other techniques such as fluorescent in situ hybridization (FISH), a culture-independent method that utilizes oligonucleotides (labeled with a fluorophore) that are specific to a string of target DNA/RNA. In the current study, the applicability of FISH was evaluated for the detection of mastitis pathogens directly in milk samples. To remove interfering lipids and proteins from mastitis milk samples prior to FISH, a previously published enzymatic treatment with savinase was evaluated. FISH was performed using oligonucleotides specific for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, and Trueperella (Arcanobacterium) pyogenes. The enzymatic pretreatment and the sensitivity of FISH were evaluated using spiked whole milk samples and mastitis milk samples with bacterial loads of less than 10(3) up to 10(8) colony-forming units (CFU)/ml. Bacteria were reliably detected in milk samples with bacterial numbers of 10(6) CFU/ml or higher. However, bacteria present in numbers below 10(6) CFU/ml were not detectable in all cases. The ability of FISH to identify mastitis-causing pathogens directly in milk samples, and therefore earlier than classical culture methods, can supplement the classic diagnostic procedures for mastitis milk samples. PMID:23632662

Gey, Annerose; Werckenthin, Christiane; Poppert, Sven; Straubinger, Reinhard K

2013-05-01

109

Complete karyotype characterization of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ hybridization, fluorescence in situ hybridization, and comparative genomic hybridization.  

PubMed

This study combines conventional cytogenetics, fluorescence in situ hybridization (FISH), multiplex-FISH and comparative genomic hybridization (CGH). In applying this multimodal approach on the human leukemia cell line K562, the chromosome composition was refined in detail and compared with data from the literature. A hypotriploid karyotype with a modal chromosome number of 67, and 21 unique marker chromosomes were identified. The classification of six markers was identical to published data and the composition of five further markers from the literature could be fully clarified for the first time. The composition of another five markers, which have been interpreted in divergent ways in different studies, were elucidated without doubt. Finally, five new markers of our study seem to have no equivalents in former studies, very likely due to limitations of conventional cytogenetics. The combinatory application of complementary techniques as shown in this study will be very useful to provide the basis of a refined genotype analysis on the chromosomal level. PMID:11248328

Naumann, S; Reutzel, D; Speicher, M; Decker, H J

2001-04-01

110

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.  

PubMed

We have developed a simple method to resolve the sex chromosome constitution in females of Lepidoptera by using a combination of genomic in situ hybridization (GISH) and fluorescence in situ hybridization with (TTAGG)( n ) telomeric probe (telomere-FISH). In pachytene configurations of sex chromosomes, GISH differentiated W heterochromatin and telomere-FISH detected the chromosome ends. With this method we showed that Antheraea yamamai has a standard system with a fully differentiated W-Z sex chromosome pair. In Orgyia antiqua, we confirmed the presence of neo-W and neo-Z chromosomes, which most probably originated by fusion of the ancestral W and Z with an autosome pair. In contrast to earlier data, Orgyia thyellina females displayed a neo-ZW(1)W(2) sex chromosome constitution. A neo-WZ(1)Z(2) trivalent was found in females of Samia cynthia subsp. indet., originating from a population in Nagano, Japan. Whereas another subspecies collected in Sapporo, Japan, and determined as S. cynthia walkeri, showed a neo-W/neo-Z bivalent similar to O. antiqua, and the subspecies S. cynthia ricini showed a Z univalent (a Z/ZZ system). The combination of GISH and telomere-FISH enabled us to acquire not only reliable information about sex chromosome constitution but also an insight into sex chromosome evolution in Lepidoptera. PMID:16021495

Yoshido, Atsuo; Marec, Frantisek; Sahara, Ken

2005-08-01

111

Comparative analysis of two thyroid tumor cell lines by fluorescence in situ hybridization and comparative genomic hybridization.  

PubMed

Tumors and tumor-derived cell lines are typically chromosomally complex and heterogeneous. These features complicate the description of their karyotype. As a first approach to the chromosomal characterization of the two near-triploid thyroid tumor cell lines, BCPAP and FTC133, the techniques of fluorescence in situ hybridization and comparative genomic hybridization were used and compared. Most of the results obtained by the two methods were in good agreement. The follicular-derived cell line FTC133 showed more extensive chromosome variation between cells than the papillary-derived cell line BCPAP. Both cell lines had significant gains in part or whole of chromosomes 1, 11, and 20 and losses in chromosomes 16, 21, and 22. BCPAP cells also had gains in chromosomes 4 and 5 and losses in chromosomes 7, 9, and 10; FTC133 cells had gains in chromosomes 6, 7, 8, 14, 15, and 19. Chromosomes 4 and 5 were the most stable in BCPAP cells; in the FTC133 cells, chromosomes 7 and 19 showed the greatest segregational stability. The results have been discussed in terms of possible karyotype evolution. Moreover, it has been possible to compare the sensitivity limits of the two techniques in the analysis of polyploid tumors. PMID:12393281

Corso, Chiara; Ulucan, Hacan; Parry, Elizabeth M; Parry, James M

2002-09-01

112

Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes  

Microsoft Academic Search

The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial

S. J. Macnaughton; A. G. O'Donnelll; T. M. Embley

1994-01-01

113

Rapid and Accurate Diagnosis of Human Intestinal Spirochetosis by Fluorescence In Situ Hybridization?  

PubMed Central

Human intestinal spirochetosis (HIS) is associated with overgrowth of the large intestine by spirochetes of the genus Brachyspira. The microbiological diagnosis of HIS is hampered by the fastidious nature and slow growth of Brachyspira spp. In clinical practice, HIS is diagnosed histopathologically, and a significant portion of cases may be missed. Fluorescence in situ hybridization (FISH) is a molecular method that allows the visualization and identification of single bacteria within tissue sections. In this study, we analyzed intestinal biopsy samples from five patients with possible HIS. All specimens yielded positive results by histopathological techniques. PCR amplification and sequencing of the 16S rRNA gene were performed. Sequences of two isolates clustered in the group of Brachyspira aalborgi, whereas in three cases, the sequences were highly similar to that of Brachyspira pilosicoli. Three phylotypes showed mismatches at distinct nucleotide positions with Brachyspira sp. sequences published previously. In addition, culture for Brachyspira was successful in three cases. On the basis of these data, we designed and evaluated a Brachyspira genus-specific 16S rRNA-directed FISH probe that detects all of the Brachyspira spp. published to date. FISH of biopsy samples resulted in strong, unequivocal signals of brush-like formations at the crypt surfaces. This technique allowed simultaneous visualization of single spirochetes and their identification as Brachyspira spp. In conclusion, FISH provides a fast and accurate technique for the visualization and identification of intestinal spirochetes in tissue sections. It therefore represents a valuable tool for routine diagnosis of HIS.

Schmiedel, Dinah; Epple, Hans-Jorg; Loddenkemper, Christoph; Ignatius, Ralf; Wagner, Jutta; Hammer, Bettina; Petrich, Annett; Stein, Harald; Gobel, Ulf B.; Schneider, Thomas; Moter, Annette

2009-01-01

114

Population Dynamics of Bifidobacterium Species in Human Feces during Raffinose Administration Monitored by Fluorescence In Situ Hybridization-Flow Cytometry  

Microsoft Academic Search

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicar- bocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at

Achmad Dinoto; Tatiana M. Marques; Kanta Sakamoto; Satoru Fukiya; Jun Watanabe; Susumu Ito; Atsushi Yokota

2006-01-01

115

Classification of multicolor fluorescence in situ hybridization (M-FISH) images with sparse representation.  

PubMed

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 on a comprehensive M-FISH database that we established, demonstrating improved performance in classification. When compared with other pixel-wise M-FISH image classifiers such as fuzzy c-means (FCM) clustering algorithms and adaptive fuzzy c-means (AFCM) clustering algorithms that we proposed earlier the current method gave the lowest classification error. In order to evaluate the performance of different SRC for M-FISH imaging analysis, three different sparse representation methods, namely, Homotopy method, Orthogonal Matching Pursuit (OMP), and Least Angle Regression (LARS), were tested and compared. Results from our statistical analysis have shown that Homotopy based method is significantly better than the other two methods. Our work indicates that sparse representations based classifiers with proper models can outperform many existing classifiers for M-FISH classification including those that we proposed before, which can significantly improve the multicolor imaging system for chromosome analysis in cancer and genetic disease diagnosis. PMID:22665392

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

2012-06-01

116

Application of flow cytometry and fluorescent in situ hybridization for assessment of exposures to airborne bacteria.  

PubMed Central

Current limitations in the methodology for enumeration and identification of airborne bacteria compromise the precision and accuracy of bioaerosol exposure assessment. In this study, flow cytometry and fluorescent in situ hybridization (FISH) were evaluated for the assessment of exposures to airborne bacteria. Laboratory-generated two-component bioaerosols in exposures chambers and complex native bioaerosols in swine barns were sampled with two types of liquid impingers (all-glass impinger-30 and May 3-stage impinger). Aliquots of collection media were processed and enumerated by a standard culture technique, microscopy, or flow cytometry after nucleic acid staining with 4',6-diamidino-2-phenylindole (DAPI) and identified taxonomically by FISH. DAPI-labeled impinger samples yielded comparable estimates of bioaerosol concentrations when enumerated by microscopy or flow cytometry. The standard culture method underestimated bioaerosol concentrations by 2 orders of magnitude when compared to microscopy or flow cytometry. In the FISH method, aliquots of collection media were incubated with a probe universally complementary to eubacteria, a probe specific for several Pseudomonas species, and a probe complementary to eubacteria for detection of nonspecific binding. With these probes, FISH allowed quantitative identification of Pseudomonas aeruginosa and Escherichia coli bioaerosols in the exposure chamber without measurable nonspecific binding. Impinger samples from the swine barn demonstrated the efficacy of the FISH method for the identification of eubacteria in a complex organic dust. This work demonstrates the potential of emerging molecular techniques to complement traditional methods of bioaerosol exposure assessment.

Lange, J L; Thorne, P S; Lynch, N

1997-01-01

117

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

PubMed Central

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.

2014-01-01

118

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

SciTech Connect

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.

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

1995-11-01

119

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

NASA Astrophysics Data System (ADS)

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.

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

2012-05-01

120

Identification of tissue contamination by polymorphic deletion probe fluorescence in situ hybridization.  

PubMed

Potential sources of error in surgical pathology include specimen misidentification, unidentified tissue, and tissue contamination of paraffin blocks and slides. Current molecular approaches to characterize unidentified or misidentified tissue include fluorescence in situ hybridization identification of sex chromosomes (XY FISH) and microsatellite analysis. Polymorphic deletion probe (PDP) FISH, a novel FISH assay based on copy number variants, can distinguish between cells and tissues from 2 individuals in situ, independent of gender. Using a panel of 3 PDPs, we compared the genotypes of potential tissue contaminants (n=19) and unidentified tissues (n=6) with patient tissues to determine the utility of PDP FISH in resolving specimen identity. XY FISH was added to increase the informative potential of the assay, and microsatellite analysis was used as a gold standard to confirm PDP FISH results. PDP FISH distinguished between putative contaminants and patient tissues in 13 of 14 cases and indicated a high likelihood of 2 tissues originating from the same source in 11 of 11 cases. The assay has a sensitivity and specificity of 86% [6/7, exact 95% confidence interval (CI): 42%, 97%] and 100% (9/9, exact 1-sided 97.5% CI: 68%, 100%), respectively, and a positive predictive value and negative predictive value of 100% (6/6, exact 1-sided 97.5% CI: 54%, 100%) and 90% (9/10, exact 95% CI: 55%, 98%), respectively. PDP FISH is an accurate and practical molecular assay for the genetic characterization of potential tissue contaminants and unidentified tissues, especially in the setting of small sample size, and permits concomitant assessment of morphology. PMID:22982889

Chiang, Sarah; Yip, Stephen; Betensky, Rebecca A; Batten, Julie M; Misdraji, Joseph; John Iafrate, A

2012-10-01

121

Estimate of true incomplete exchanges using fluorescence in situ hybridization with telomere probes  

NASA Technical Reports Server (NTRS)

PURPOSE: To study the frequency of true incomplete exchanges in radiation-induced chromosome aberrations. MATERIALS AND METHODS: Human lymphocytes were exposed to 2 Gy and 5 Gy of gamma-rays. Chromosome aberrations were studied using the fluorescence in situ hybridization (FISH) technique with whole chromosome-specific probes, together with human telomere probes. Chromosomes 2 and 4 were chosen in the present study. RESULTS: The percentage of incomplete exchanges was 27% when telomere signals were not considered. After excluding false incomplete exchanges identified by the telomere signals, the percentage of incomplete exchanges decreased to 11%. Since telomere signals appear on about 82% of the telomeres, the percentage of true incomplete exchanges should be even lower and was estimated to be 3%. This percentage was similar for chromosomes 2 and 4 and for doses of both 2 Gy and 5 Gy. CONCLUSIONS: The percentage of true incomplete exchanges is significantly lower in gamma-irradiated human lymphocytes than the frequencies reported in the literature.

Wu, H.; George, K.; Yang, T. C.

1998-01-01

122

Interphase fluorescence in situ hybridization signal detection by computing intensity variance along the optical axis  

NASA Astrophysics Data System (ADS)

Fluorescence in situ Hybridization technology is a commonly used tool to detect chromosome aberrations, which are often pathologically significant. Since manual FISH analysis is a tedious and time-consuming procedure, reliable and robust automated image acquisition and analysis are in demand. Under high magnification objective lenses such as 60x and 100x, the depth of field will often be too small and the FISH probes may not always lie in the same focal plane. A statistical variance based automated FISH analysis method is developed in order to address this problem. On a stack of slices at consecutive image planes with a step size d, the statistical variance alone the z-axis is calculated to form a 2-D matrix. Since pixels shift dramatically to high intensity at FISH probe location, the probes will manifest high peak values in the matrix. A computer-aided detection scheme based on top-hat transform is applied to the matrix to detect FISH probe signals. This study demonstrates a simple and robust method for FISH probe detection as well as a way of 2- D representation of 3-D data.

Li, Zheng; Zheng, Bin; Ren, Liqiang; Liu, Hong

2014-02-01

123

Does polyomavirus infection interfere with bladder cancer fluorescence in situ hybridization?  

PubMed

Urine cytology is a proven and widely used screening tool for the detection of urothelial carcinoma. However, morphologic features of polyomavirus infected cells, characterized by nuclear inclusions (decoy cells) are a known source of diagnostic confusion with malignancy. Fluorescence in situ hybridization (FISH) is now routinely used to support the cytological diagnosis of urothelial carcinoma and monitor for recurrence. We sought to determine whether polyomavirus infection could result in positive FISH results (aneuploidy). This study deals with retrospective study of 100 polyomavirus-infected urine samples from patients with no history of urothelial carcinoma or organ transplantation. All cases were stained with Papanicolaou and acid hematoxylin stain. One slide from each sample was de-stained and FISH was performed using chromosome enumeration probes 3, 7, 17, and locus-specific probe 9p21. Adequate cells for FISH analysis (25 cells) were present in 81 cases; 19 cases were insufficient due to loss of cells during de-staining and FISH preparation process. All polyomavirus-infected cells (decoy cells) exhibited a normal chromosome pattern. Four cases were FISH positive, but there were no positive decoy cells. Decoy cells did not exhibit aneuploidy by FISH. The presence of decoy cells does not exclude the possibility of concurrent urothelial carcinoma. Acid hematoxylin stain appeared to supplement the Papanicolou stain in identifying and confirming the presence of polyomavirus infection. PMID:24006232

Hossain, Deloar; Hull, David; Kalantarpour, Fatemeh; Maitlen, Rebecca; Qian, Junqi; Bostwick, David G

2014-03-01

124

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

SciTech Connect

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.

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

1996-06-14

125

Microfluidic fluorescence in situ hybridization and flow cytometry (?FlowFISH).  

PubMed

We describe an integrated microfluidic device (?FlowFISH) capable of performing 16S rRNA fluorescence in situ hybridization (FISH) followed by flow cytometric detection for identifying bacteria in natural microbial communities. The device was used for detection of species involved in bioremediation of Cr(vi) and other metals in groundwater samples from a highly-contaminated environmental site (Hanford, WA, USA). The ?FlowFISH seamlessly integrates two components: a hybridization chamber formed between two photopolymerized membranes, where cells and probes are electrophoretically loaded, incubated and washed, and a downstream cross structure for electrokinetically focusing cells into a single-file flow for flow cytometry analysis. The device is capable of analyzing a wide variety of bacteria including aerobic, facultative and anaerobic bacteria and was initially tested and validated using cultured microbes, including Escherichia coli, as well as two strains isolated from Hanford site: Desulfovibrio vulgaris strain RCH1, and Pseudomonas sp.strain RCH2 that are involved in Cr(vi) reduction and immobilization. Combined labeling and detection efficiencies of 74-97% were observed in experiments with simple mixtures of cultured cells, confirming specific labeling. Results obtained were in excellent agreement with those obtained by conventional flow cytometry confirming the accuracy of ?FlowFISH. Finally, the device was used for analyzing water samples collected on different dates from the Hanford site. We were able to monitor the numbers of Pseudomonas sp. with only 100-200 cells loaded into the microchip. The ?FlowFISH approach provides an automated platform for quantitative detection of microbial cells from complex samples, and is ideally suited for analysis of precious samples with low cell numbers such as those found at extreme environmental niches, bioremediation sites, and the human microbiome. PMID:21755095

Liu, Peng; Meagher, Robert J; Light, Yooli K; Yilmaz, Suzan; Chakraborty, Romy; Arkin, Adam P; Hazen, Terry C; Singh, Anup K

2011-08-21

126

Fifty probands with extra structurally abnormal chromosomes characterized by fluorescence in situ hybridization  

SciTech Connect

Extra structurally abnormal chromosomes (ESACs) are small supernumerary chromosomes often associated with developmental abnormalities and malformations. We present 50 probands with ESACs characterized by fluorescence in situ hybridization using centromere-specific probes and chromosome-specific libraries. ESAC-specific libraries were constructed by flow sorting and subsequent amplification by DOP-PCR. Using such ESAC-specific libraries we were able to outline the chromosome regions involved. Twenty-three of the 50 ESACs were inverted duplications of chromosome 15 (inv dup(15)), including patients with normal phenotypes and others with similar clinical symptoms. These 2 groups differed in size and shape of the inv dup(15). Patients with a large inv dup(15), which included the Prader-Willi region, had a high risk of abnormality, whereas patients with a small inv dup(15), not including the Prader-Willi region, were normal. ESACs derived from chromosomes 13 or 21 appeared to have a low risk of abnormality, while one out of 3 patients with an ESAC derived from chromosome 14 had discrete symptoms. One out of 3 patients with an ESAC derived from chromosome 22 had severe anomalies, corresponding to some of the manifestations of the cat eye syndrome. Small extra ring chromosomes of autosomal origin and ESACs identified as i(12p) or i(18p) were all associated with a high risk of abnormality. 42 refs., 2 figs., 2 tabs.

Blennow, E.; Telenius, H.; Nordenskjoeld, M. [Karolinska Hospital, Stockholm (Sweden)] [and others

1995-01-02

127

An extended fluorescence in situ hybridization approach for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing cytology preparations.  

PubMed

The cytological diagnosis of cholangiocarcinoma has been significantly aided by applying a 4-probe fluorescence in situ hybridization system on endoscopic retrograde cholangiopancreatography brushing smears, aiming mainly at the detection of hyperdiploidy. However, this approach adds little to our understanding of the genetic background of the disease. With the prospect of obtaining additional data on chromosomal aberrations, we have extended the fluorescence in situ hybridization study, with the application of 4 independent 2-probe systems in 35 patients with documented cholangiocarcinoma. Fluorescence in situ hybridization assays were performed on endoscopic retrograde cholangiopancreatography brushing smears, with probes for the 7q31, 11q13 (CCND1), 17p53 (TP53), and 9p21 (INK4 locus) bands, together with the respective centromeric probe. Hyperdiploidy, involving at least 2 of the 4 chromosomes targeted, was found in 31 patients. 17p13 deletion was detected in 3, and 9p21 deletion, in 5 of the hyperdiploid cases, with the 2 aberrations concurrent in 1. CCND1 amplification was found in 1 case as the sole abnormality and in another together with hyperdiploidy, but in apparently unrelated clones. This work indicates that interphase fluorescence in situ hybridization is a practical and useful tool for the cytogenetic study of cholangiocarcinoma on endoscopic retrograde cholangiopancreatography brushing smears, which is often the only available tissue specimen of the tumor. Apart from hyperdiploidy, it provides additional data on the genetic profile of cholangiocarcinoma, especially regarding structural chromosomal aberrations and clonal diversity. This line of investigation may prove useful in the delineation of oncogenesis and the interpretation of the diverse clinical features of the disease. PMID:23845469

Vasilieva, Larisa E; Papadhimitriou, Stefanos I; Alexopoulou, Alexandra; Pavlidis, Dimitris; Kostopoulos, Ioannis; Georgiakaki, Maria; Xinopoulos, Dimitrios; Romanos, Andreas; Dourakis, Spyridon P

2013-10-01

128

Fluorescence in situ hybridization investigation of potentially pathogenic bacteria involved in neonatal porcine diarrhea  

PubMed Central

Background Neonatal diarrhea is a multifactorial condition commonly present on pig farms and leads to economic losses due to increased morbidity and mortality of piglets. Immature immune system and lack of fully established microbiota at birth predispose neonatal piglets to infection with enteric pathogens. The microorganisms that for decades have been associated with enteritis and diarrhea in suckling piglets are: rotavirus A, coronavirus, enterotoxigenic Escherichia coli (ETEC), Clostridium perfringens type C, Cryptosporidium spp., Giardia spp., Cystoisospora suis and Strongyloides ransomi. However, in recent years, the pig industry has experienced an increased number of neonatal diarrhea cases in which the above mentioned pathogens are no longer detected. Potentially pathogenic bacteria have recently received focus in the research on the possible etiology of neonatal diarrhea not caused by common pathogens. The primary aim of this study was to investigate the role of E. coli, Enterococcus spp., C. perfringens and C. difficile in the pathogenesis of neonatal porcine diarrhea with no established casual agents. Fluorescence in situ hybridization with oligonucleotide probes was applied on the fixed intestinal tissue samples from 51 diarrheic and 50 non-diarrheic piglets collected from four Danish farms during outbreaks of neonatal diarrhea not caused by well-known enteric pathogens. Furthermore, an association between the presence of these bacteria and histological lesions was evaluated. Results The prevalence of fluorescence signals specific for E. coli, C. perfringens and C. difficile was similar in both groups of piglets. However, Enterococcus spp. was primarily detected in the diarrheic piglets. Furthermore, adherent bacteria were detected in 37 % diarrheic and 14 % non-diarrheic piglets. These bacteria were identified as E. coli and Enterococcus spp. and their presence in the intestinal mucosa was associated with histopathological changes. Conclusions The results of this study showed that simultaneous colonization of the intestinal mucosa by adherent non-ETEC E. coli and Enterococcus spp. can be involved in the pathogenesis of neonatal porcine diarrhea. These bacteria should be considered in diagnosis of diarrhea in piglets, when detection of common, well-known enteric agents is unsuccessful.

2014-01-01

129

Fluorescence in situ hybridization on paraffin-embedded abortion material as a means of retrospective chromosome analysis  

Microsoft Academic Search

A fluorescence in situ hybridization (FISH) procedure was used to detect chromosome abnormalities in archival abortion material. Nuclei were isolated from 50-µm-thick tissue blocks from 18 selected and karyotyped abortions. Five probes for repetitive centromeric sequences of chromosomes 1, 16, 18, X and Y were used. For each chromosome, at least 200 nuclei were scored blindly, i.e. without knowledge of

Gesina van Lijnschoten; Jozefa Albrechts; Monique Vallinga; Anton H. N. Hopman; Jan W. Arends; Joseph P. M. Geraedts

1994-01-01

130

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

Microsoft Academic Search

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).

Ramesh C. Juyal; P. I. Patel; F. Greenberg; James R. Lupski; Barbara J. Trask; Ger van den Engh; Elizabeth A. Lindsay; Heather Christy; Ken-Shiung Chen; Antonio Baldini; Lisa G. Shaffer

1995-01-01

131

Simple Adhesive-Tape-Based Sampling of Tomato Surfaces Combined with Rapid Fluorescence In Situ Hybridization for Salmonella Detection?  

PubMed Central

A simple adhesive-tape-based method for sampling of tomato surfaces was combined with fluorescence in situ hybridization for rapid culture-independent detection of Salmonella strains. Tapes could also be placed face-down on selective agar for on-tape enrichment of captured Salmonella cells. Overlay of cell-charged tapes with small volumes of liquid enrichment media enabled subsequent detection of tape-captured Salmonella via flow cytometry.

Bisha, Bledar; Brehm-Stecher, Byron F.

2009-01-01

132

Localization of human CREBBP (CREB Binding Protein) to 16p13.3 by fluorescence in situ hybridization  

Microsoft Academic Search

Human and mouse cDNAs for a 265-kDa protein which binds specifically to phosphorylated cAMP-response element binding protein (CREB), have recently been isolated. A cDNA probe for the mouse CREB binding protein was used to localize this gene (CREBBP) to human chromosome 16p13.3 by fluorescence in situ hybridization (FISH).Copyright © 1995 S. Karger AG, Basel

X.-N. Chen; J. R. Korenberg

1995-01-01

133

Detection of Gallibacterium spp. in Chickens by Fluorescent 16S rRNA In Situ Hybridization  

PubMed Central

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, “Actinobacillus salpingitidis,” and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens.

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-01-01

134

Detection of Gallibacterium spp. in chickens by fluorescent 16S rRNA in situ hybridization.  

PubMed

Gallibacterium has recently been included as a new genus of the family Pasteurellaceae Pohl 1981, which encompasses bacteria previously reported as Pasteurella anatis, "Actinobacillus salpingitidis," and avian Pasteurella haemolytica-like organisms. So far, identification has exclusively relied on phenotypic characterization. We present a method based on a cyanine dye 3.18-labeled in situ hybridization probe targeting 16S rRNA to allow specific detection of bacteria belonging to the genus Gallibacterium. The probe, GAN850, showed no cross-reactivity to 25 other poultry-associated bacterial species, including members of the families Pasteurellaceae, Enterobacteriaceae, and Flavobacteriaceae, when cross-reactivities were evaluated by whole-cell hybridization. The probe was further evaluated by hybridization to formalin-fixed spleen and liver tissues from experimentally infected chickens, in which it proved to be useful for the detection of Gallibacterium. Additionally, determination of the spatial distribution and the host cell affiliation of Gallibacterium at various times during the infection process was possible. In conclusion, the in situ hybridization technique described may be of use as a diagnostic tool as well as for studies to elucidate the pathogenesis of Gallibacterium infections in chickens. PMID:14605154

Bojesen, Anders Miki; Christensen, Henrik; Nielsen, Ole Lerberg; Olsen, John Elmerdahl; Bisgaard, Magne

2003-11-01

135

Sample preparations are important for fluorescence in situ hybridization of cells biopsied from preimplantation embryos.  

PubMed

Summary Fluorescence in situ hybridization (FISH) is a cytogenetic technology used to detect chromosomal abnormalities in preimplantation human embryos. However, its efficiency is not stable due to improper sample preparation. The present study was designed to modify the current sample preparation technique and then to evaluate its efficiency in human preimplantation genetic diagnosis (PGD). Day 3 cleavage embryos as well as day 5 and 6 blastocysts were biopsied by mechanical aspiration method. In the present study, two methods were used for sample preparation of the biopsied cells. Method I was the traditional method, in which each blastomere was placed in a hypotonic solution for 5 min and then fixed on glass slides. The slides were kept at room temperature before the FISH procedures. Method II was a modified method, in which all blastomeres were placed individually in hypotonic solution drops covered by oil for at least 5 min and then fixed on slides with 0.1% Tween/HCl. After fixation, the slides were kept at -20°C for at least 30 min before the FISH procedures. The two methods were compared in terms of time consumption and proportions of blastomeres with FISH signals. In total, 329 blastomeres from day 3 embryos were fixed by Method I with an average fixation time of 8-10 min for each blastomere. By contrast, with Method II, 362 blastomeres were fixed and the average time was 3-4 min for each blastomere. After FISH, more nuclei had signals with Method II (97.2%) than with Method I (86.9%). All cells that were biopsied from blastocysts and prepared with Method II had FISH signals. However, Method I was not suitable for the fixation of multiple cells biopsied from blastocysts as cells were not traceable during the fixation. The present study indicates that proper sample preparation is critical for obtaining FISH signals in cells biopsied from preimplantation human embryos; hence these modifications can increase the efficiency of human PGD. PMID:23331574

Li, Lifei; Zhang, Xuehong; Wang, Weihua

2014-08-01

136

A pre-breeding screening program for transgenic boars based on fluorescence in situ hybridization assay.  

PubMed

For efficient transgenic herd expansion, only the transgenic animals that possess the ability to transmit transgene into next generation are considered for breeding. However, for transgenic pig, practically lacking a pre-breeding screening program, time, labor and money is always wasted to maintain non-transgenic pigs, low or null transgenic transmission pigs and the related fruitless gestations. Developing a pre-breeding screening program would make the transgenic herd expansion more economical and efficient. In this technical report, we proposed a three-step pre-breeding screening program for transgenic boars simply through combining the fluorescence in situ hybridization (FISH) assay with the common pre-breeding screening workflow. In the first step of screening, combined with general transgenic phenotype analysis, FISH is used to identify transgenic boars. In the second step of screening, combined with conventional semen test, FISH is used to detect transgenic sperm, thus to identify the individuals producing high quality semen and transgenic sperm. In the third step of screening, FISH is used to assess the in vitro fertilization embryos, thus finally to identify the individuals with the ability to produce transgenic embryos. By this three-step screening, the non-transgenic boars and boars with no ability to produce transgenic sperm or transgenic embryos would be eliminated; therefore only those boars could produce transgenic offspring are maintained and used for breeding and herd expansion. It is the first time a systematic pre-breeding screening program is proposed for transgenic pigs. This program might also be applied in other transgenic large animals, and provide an economical and efficient strategy for herd expansion. PMID:24788205

Bou, Gerelchimeg; Sun, Mingju; Lv, Ming; Zhu, Jiang; Li, Hui; Wang, Juan; Li, Lu; Liu, Zhongfeng; Zheng, Zhong; He, Wenteng; Kong, Qingran; Liu, Zhonghua

2014-08-01

137

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy  

PubMed Central

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

1994-01-01

138

Fluorescent In Situ Hybridization: A New Tool for the Direct Identification and Detection of F. psychrophilum  

PubMed Central

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.

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

2012-01-01

139

Enzymatic production of single-stranded DNA as a target for fluorescence in situ hybridization  

Microsoft Academic Search

This study demonstrates that Exonuclease III (Exo III) can be used to produce sufficient single-stranded (ss)DNA in chromosomes and cells to allow in situ hybridization. In this study, all of the probes were modified with biotin and the probe binding was visualized with fluorescein-labeled avidin. Exo III digestion starting at naturally occurring breaks in methanol-acetic acid preparations produced enough ssDNA

H. van Dekken; D. Pinkel; J. Mullikin; J. W. Gray

1988-01-01

140

Effect of fixation period on HER2/neu gene amplification detected by fluorescence in situ hybridization in invasive breast carcinoma.  

PubMed

This study investigated if formalin fixation duration affects HER2/neu gene amplification detection by fluorescence in situ hybridization (FISH) in breast cancer. Tumor tissues from 35 cases were divided into three groups and subjected to two formalin fixation protocols per group (12 hr, 27 hr in the first; 2 hr, 17.5 hr in the second; 28.5 hr, 541 hr in the third) before FISH analysis. There was no significant difference in FISH signal detection between the two different fixation protocols in the first two groups. In the third, no signal was detected in 4/6 cases fixed for an extended duration. PMID:12486093

Selvarajan, Sathiyamoorthy; Bay, Boon-Huat; Choo, Andrew; Chuah, Khoon-Leong; Sivaswaren, Christina Rudduck; Tien, Sim-Leng; Wong, Chow-Yin; Tan, Puay-Hoon

2002-12-01

141

Use of fluorescence in situ hybridization to clarify a complex chromosomal rearrangement in a child with multiple congenital anomalies  

SciTech Connect

A child with multiple congenital anomalies was referred for cytogenetic evaluation. G-banded analysis showed a complex chromosome rearrangement involving 6 different chromosomes and 10 breakpoints. Fluorescence in situ hybridization (FISH) using whole chromosome painting probes and repetitive sequence probes was performed. In most cases the painting probes alone helped to clarify the G-banded results. However, in one instance, where the terminal band of the long arm of chromosome 1 was involved, the use of a telomeric probe was essential in defining the rearrangement. 13 refs., 3 figs.

Spikes, A.S.; Hegmann, K.; Smith, J.L. [Baylor College of Medicine, Houston, TX (United States)] [and others

1995-05-22

142

Comparative mapping of seven genes in mouse, rat and Chinese hamster chromosomes by fluorescence in situ hybridization  

Microsoft Academic Search

By fluorescence in situ hybridization (FISH) using mouse probes, we assigned homologues for cathepsin E (Ctse), protocadherin 10 (Pcdh10, alias OL-protocadherin, Ol-pc), protocadherin 13 (Pcdh13, alias protocadherin 2c, Pcdh2c), neuroglycan C (Cspg5) and myosin X (Myo10) genes to rat chromosomes (RNO) 13q13, 2q24?q25, 18p12?p11, 8q32.1 and 2q22.1?q22.3, respectively. Similarly, homologues for mouse Ctse, Pcdh13, Cspg5 and Myo10 genes and homologues

T. Ono; S. Hirano; S. Yonezawa; S. Aono; M. Osaki; S. Masaki; S. Yamashita; T. Tsukasaki; A. Oohira; S. T. Suzuki; S. Sonta

2000-01-01

143

Specific detection of green sulfur bacteria by in situ hybridization with a fluorescently labeled oligonucleotide probe.  

PubMed

An oligodeoxynucleotide probe (GSB-532) specific for green sulfur bacteria was developed. Highly stringent hybridization conditions were established using whole cells of Chlorobium limicola DSM249 immobilized on glass slides. At a formamide concentration of 10%, the optimum specificity was reached at 47 degrees C. When a conventional fixation procedure was used, a conspicuous autofluorescence developed within the cells. This autofluorescence was due to the liberation of bacteriochlorophyll by the detergent Triton X-100 and a subsequent conversion to bacteriophenophytin and related compounds. The signal-to-noise ratio could be increased by a final dehydration of the samples with methanol. Finally, the method was adapted to the hybridization of natural samples collected on polycarbonate membrane filters. In situ hybridization of pure cultures, various enrichments, and natural samples from the chemocline of a freshwater lake confirmed that probe GSB-532 hybridized exclusively to cells of green sulfur bacteria. Our protocol allows the highly specific detection of green sulfur bacteria in water samples and a rapid screening of natural bacterial communities. Employing probe GSB-532, the phylogenetic affiliation of the epibionts in "Chlorochromatium aggregatum" and "Pelochromatium roseum" could be demonstrated for the first time. PMID:10339808

Tuschak, C; Glaeser, J; Overmann, J

1999-03-01

144

A high-resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolor fluorescence in situ hybridization  

Microsoft Academic Search

A molecular cytogenetic map of Chinese cabbage ( Brassica rapa ssp. pekinensis, 2 n=20) was constructed based on the 4?-6-diamino-2-phenylindole dihydrochloride-stained mitotic metaphase and pachytene chromosomes and multicolor fluorescence in situ hybridization (McFISH), using three repetitive DNA sequences, 5S rDNA, 45S rDNA, and C11-350H. The lengths of mitotic metaphase chromosomes ranged from 1.46 ?m to 3.30 ?m. Five 45S and three 5S

Dal-Hoe Koo; Prikshit Plaha; Yong Pyo Lim; Yoonkang Hur; Jae-Wook Bang

2004-01-01

145

Enzymatic Permeabilization of the Thecate Dinoflagellate Alexandrium minutum (Dinophyceae) Yields Detection of Intracellularly Associated Bacteria via Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization  

Microsoft Academic Search

The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes.

L. Palacios; I. Marin

2008-01-01

146

Enzymatic Permeabilization of the Thecate Dinoflagellate Alexandrium minutum (Dinophyceae) Yields Detection of Intracellularly Associated Bacteria via Catalyzed Reporter Deposition-Fluorescence In Situ Hybridization?  

PubMed Central

The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes.

Palacios, Lucia; Marin, Irma

2008-01-01

147

Enzymatic permeabilization of the thecate dinoflagellate Alexandrium minutum (Dinophyceae) yields detection of intracellularly associated bacteria via catalyzed reporter deposition-fluorescence in situ hybridization.  

PubMed

The enzymatic permeabilization procedure described here allows the detection of intracellular bacteria in the thecate dinoflagellate Alexandrium minutum by using catalyzed reporter deposition-fluorescence in situ hybridization. The combined use of propidium iodide and calcofluor for confocal laser scanning microscopy, together with general and specific fluorescent bacterial probes, demonstrated the intracellular presence of bacteria, including members of the phylum Bacteroidetes. PMID:18263745

Palacios, Lucía; Marín, Irma

2008-04-01

148

Development of a fluorescence in situ hybridization protocol for the identification of micro-organisms associated with wastewater particles and flocs  

Microsoft Academic Search

Fluorescence in situ hybridization (FISH) provides a unique tool to study micro-organisms associated with particles and flocs. FISH enables visual examination of micro-organisms while they are structurally intact and associated with particles. However, application of FISH to wastewater and sludge samples presents a specific set of problems. Wastewater samples generate high background fluorescence due to their organic and inorganic content

Banu Örmeci; Karl G. Linden

2008-01-01

149

Combined interphase fluorescence in situ hybridization elucidates the genetic heterogeneity of T-cell acute lymphoblastic leukemia in adults  

PubMed Central

Background Molecular lesions in T-cell acute lymphoblastic leukemias affect regulators of cell cycle, proliferation, differentiation, survival and apoptosis in multi-step pathogenic pathways. Full genetic characterization is needed to identify events concurring in the development of these leukemias. Design and Methods We designed a combined interphase fluorescence in situ hybridization strategy to study 25 oncogenes/tumor suppressor genes in T-cell acute lymphoblastic leukemias and applied it in 23 adult patients for whom immunophenotyping, karyotyping, molecular studies, and gene expression profiling data were available. The results were confirmed and integrated with those of multiplex-polymerase chain reaction analysis and gene expression profiling in another 129 adults with T-cell acute lymphoblastic leukemias. Results The combined hybridization was abnormal in 21/23 patients (91%), and revealed multiple genomic changes in 13 (56%). It found abnormalities known to be associated with T-cell acute lymphoblastic leukemias, i.e. CDKN2A-B/9p21 and GRIK2/6q16 deletions, TCR and TLX3 rearrangements, SIL-TAL1, CALM-AF10, MLL-translocations, del(17)(q12)/NF1 and other cryptic genomic imbalances, i.e. 9q34, 11p, 12p, and 17q11 duplication, del(5)(q35), del(7)(q34), del(9)(q34), del(12)(p13), and del(14)(q11). It revealed new cytogenetic mechanisms for TCRB-driven oncogene activation and C-MYB duplication. In two cases with cryptic del(9)(q34), fluorescence in situ hybridization and reverse transcriptase polymerase chain reaction detected the TAF_INUP214 fusion and gene expression profiling identified a signature characterized by HOXA and NUP214 upregulation and TAF_I, FNBP1, C9orf78, and USP20 down-regulation. Multiplex-polymerase chain reaction analysis and gene expression profiling of 129 further cases found five additional cases of TAF_I-NUP214-positive T-cell acute lymphoblastic leukemia. Conclusions Our combined interphase fluorescence in situ hybridization strategy greatly improved the detection of genetic abnormalities in adult T-cell acute lymphoblastic leukemias. It identified new tumor suppressor genes/oncogenes involved in leukemogenesis and highlighted concurrent involvement of genes. The estimated incidence of TAF_I-NUP214, a new recurrent fusion in adult T-cell acute lymphoblastic leukemias, was 4.6% (7/152).

Gorello, Paolo; La Starza, Roberta; Varasano, Emanuela; Chiaretti, Sabina; Elia, Loredana; Pierini, Valentina; Barba, Gianluca; Brandimarte, Lucia; Crescenzi, Barbara; Vitale, Antonella; Messina, Monica; Grammatico, Sara; Mancini, Marco; Matteucci, Caterina; Bardi, Antonella; Guarini, Anna; Martelli, Massimo Fabrizio; Foa, Robin; Mecucci, Cristina

2010-01-01

150

Fluorescence in situ Hybridization With Alu and L1 Polymerase Chain Reaction Probes for Rapid Characterization of Human Chromosomes in Hybrid Cell Lines  

Microsoft Academic Search

Human-rodent hybrid cell lines have been analyzed with regard to their human DNA content by using various DNA probe sets, derived from the hybrids, for in situ hybridization to normal human metaphase chromosome spreads. Total genomic hybrid DNA was compared with probe sets of hybrid DNA that were highly enriched in human sequences. The latter probes were obtained by amplification

Peter Lichter; Susan A. Ledbetter; David H. Ledbetter; David C. Ward

1990-01-01

151

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

PubMed Central

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 ameliorated by technical modifications such as catalyzed reporter deposition (CARD)-FISH, but the minimal numbers of rRNA copies needed to obtain a visible signal of a microbial cell after FISH or CARD-FISH have not been determined previously. In this study, a novel competitive FISH approach was developed and used to determine, based on a thermodynamic model of probe competition, the numbers of 16S rRNA copies per cell required to detect bacteria by FISH and CARD-FISH with oligonucleotide probes in mixed pure cultures and in activated sludge. The detection limits of conventional FISH with Cy3-labeled probe EUB338-I were found to be 370 ± 45 16S rRNA molecules per cell for Escherichia coli hybridized on glass microscope slides and 1,400 ± 170 16S rRNA copies per E. coli cell in activated sludge. For CARD-FISH the values ranged from 8.9 ± 1.5 to 14 ± 2 and from 36 ± 6 to 54 ± 7 16S rRNA molecules per cell, respectively, indicating that the sensitivity of CARD-FISH was 26- to 41-fold higher than that of conventional FISH. These results suggest that optimized FISH protocols using oligonucleotide probes could be suitable for more recent applications of FISH (for example, to detect mRNA in situ in microbial cells).

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

2008-01-01

152

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

SciTech Connect

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.

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

153

Permeabilization of mycolic-acid-containing actinomycetes for in situ hybridization with fluorescently labelled oligonucleotide probes.  

PubMed

The application of whole-cell hybridization using labelled oligonucleotide probes in microbial systematics and ecology is limited by difficulties in permeabilizing many Gram-positive organisms. In this investigation paraformaldehyde treatment, acid methanolysis and acid hydrolysis were evaluated as a means of permeabilizing mycolic-acid-containing actinomycetes prior to hybridization with a fluorescently labelled oligonucleotide probe designed to bind to a conserved sequence of bacterial 16S rRNA. Methods were evaluated on stationary-phase cultures of Gordona bronchialis, Mycobacterium fortuitum, Nocardia asteroides, N. brasiliensis, Rhodococcus equi, R. erythropolis, R. fascians, R. rhodochrous and Tsukamurella paurometabola, none of which could be probed following 4% (w/v) paraformaldehyde fixation. For comparison and to test the general applicability of mild acid pretreatments, Bacillus subtilis, Lactobacillus plantarum, Escherichia coli and Pseudomonas putida were also studied. The data showed that most of the mycolic-acid-containing organisms were successfully permeabilized by mild acid hydrolysis in 1 M HCl at 37 degrees C. Cells were treated for different lengths of time. In general, the mycolic-acid-containing organisms required between 30 and 50 min hydrolysis, whereas B. subtilis, E. coli and P. putida were rendered permeable in only 10 min. Interestingly, L. plantarum could not be permeabilized using acid hydrolysis even after 60 min exposure to 1 M HCl. PMID:8000549

Macnaughton, S J; O'Donnell, A G; Embley, T M

1994-10-01

154

Fluorescence in situ hybridizations (FISH) for the localization of viruses and endosymbiotic bacteria in plant and insect tissues.  

PubMed

Fluorescence in situ hybridization (FISH) is a name given to a variety of techniques commonly used for visualizing gene transcripts in eukaryotic cells and can be further modified to visualize other components in the cell such as infection with viruses and bacteria. Spatial localization and visualization of viruses and bacteria during the infection process is an essential step that complements expression profiling experiments such as microarrays and RNAseq in response to different stimuli. Understanding the spatiotemporal infections with these agents complements biological experiments aimed at understanding their interaction with cellular components. Several techniques for visualizing viruses and bacteria such as reporter gene systems or immunohistochemical methods are time-consuming, and some are limited to work with model organisms and involve complex methodologies. FISH that targets RNA or DNA species in the cell is a relatively easy and fast method for studying spatiotemporal localization of genes and for diagnostic purposes. This method can be robust and relatively easy to implement when the protocols employ short hybridizing, commercially-purchased probes, which are not expensive. This is particularly robust when sample preparation, fixation, hybridization, and microscopic visualization do not involve complex steps. Here we describe a protocol for localization of bacteria and viruses in insect and plant tissues. The method is based on simple preparation, fixation, and hybridization of insect whole mounts and dissected organs or hand-made plant sections, with 20 base pairs short DNA probes conjugated to fluorescent dyes on their 5' or 3' ends. This protocol has been successfully applied to a number of insect and plant tissues, and can be used to analyze expression of mRNAs or other RNA or DNA species in the cell. PMID:24637389

Kliot, Adi; Kontsedalov, Svetlana; Lebedev, Galina; Brumin, Marina; Cathrin, Pakkianathan Britto; Marubayashi, Julio Massaharu; Skaljac, Marisa; Belausov, Eduard; Czosnek, Henryk; Ghanim, Murad

2014-01-01

155

Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization.  

PubMed

We evaluated HER2/neu gene amplification by fluorescence in situ hybridization (FISH) in archival paraffin-embedded breast cancer tissues. Tumors from 63 human invasive breast cancers were categorized into two groups depending on whether the paraffin-embedded tissue blocks had been stored for more or less than 12 months duration. These were subjected to routine and modified FISH protocols. As microwave oven formalin fixation of tissues was carried out in the majority of the older archived specimens, the effect of this fixation method was also analyzed. FISH signals were obtained in all 13 archival specimens stored for less than 12 months. However, in 50 specimens stored for more than 12 months duration, the procedure was successful in only 10 specimens (20%), for which the pretreatment procedure had to be individually optimized for each specimen. There was no significant difference in the detection of FISH signals between microwave oven and routinely fixed specimens. PMID:12898274

Selvarajan, Sathiyamoorthy; Bay, Boon-Huat; Mamat, Shalawati Binte; Choo, Andrew; Chuah, Khoon-Leong; Sivaswaren, Christina Rudduck; Tien, Sim-Leng; Wong, Chow-Yin; Tan, Puay-Hoon

2003-09-01

156

The role of immunohistochemistry and fluorescence in situ hybridization for HER2/neu in assessing the prognosis of breast cancer.  

PubMed

Immunohistochemistry has been used in the past to identify overexpression of HER-2/neu protein on the cell membrane of breast cancer cells in fixed tissues. Most studies that have attempted to find an association between HER-2/neu expression and a poor prognosis have relied on this technique, which has intrinsic variability due to the antibody used and the degradation of surface proteins by fixation. Recent studies with fluorescence in situ hybridization have tended to confirm the purported association, showing that HER-2/neu overexpression causes a more aggressive, less responsive breast cancer. In many studies, the amplification of the HER-2/neu gene was the most important variable determining outcome, independent of other variables, such as tumor size and estrogen receptor status. PMID:10482202

Mitchell, M S; Press, M F

1999-08-01

157

Visualization and Enumeration of Marine Planktonic Archaea and Bacteria by Using Polyribonucleotide Probes and Fluorescent In Situ Hybridization  

PubMed Central

Fluorescent in situ hybridization (FISH) using rRNA-specific oligonucleotide probes has emerged as a popular technique for identifying individual microbial cells. In natural samples, however, the signal derived from fluor-labeled oligonucleotide probes often is undetectable above background fluorescence in many cells. To circumvent this difficulty, we applied fluorochrome-labeled polyribonucleotide probes to identify and enumerate marine planktonic archaea and bacteria. The approach greatly enhanced the sensitivity and applicability of FISH with seawater samples, allowing confident identification and enumeration of planktonic cells to ocean depths of 3,400 m. Quantitative whole-cell hybridization experiments using these probes accounted for 90 to 100% of the total 4?,6-diamidino-2-phenylindole (DAPI)-stained cells in most samples. As predicted in a previous study (R. Massana, A. E. Murray, C. M. Preston, and E. F. DeLong, Appl. Environ. Microbiol. 63:50–56, 1997), group I and II marine archaea predominate in different zones in the water column, with maximal cell densities of 105/ml. The high cell densities of archaea, extending from surface waters to abyssal depths, suggest that they represent a large and significant fraction of the total picoplankton biomass in coastal ocean waters. The data also show that the vast majority of planktonic prokaryotes contain significant numbers of ribosomes, rendering them easily detectable with polyribonucleotide probes. These results imply that the majority of planktonic cells visualized by DAPI do not represent lysed cells or “ghosts,” as was suggested in a previous report.

DeLong, Edward F.; Taylor, Lance Trent; Marsh, Terence L.; Preston, Christina M.

1999-01-01

158

Combined fluorescent in situ hybridization for detection of microRNAs and immunofluorescent labeling for cell-type markers  

PubMed Central

Identification of the cell type of origin for normal or aberrant gene expression is critical for many studies, and poses a significant problem for some regulatory RNAs such as microRNAs. MicroRNAs are small non-coding RNAs that regulate cellular function by targeting specific mRNAs and reducing the level of their protein product. Aberrant expression of miRNAs in cell-types where they are not normally expressed occurs in several disease conditions. Therefore, it is important to determine not only the expression level of microRNAs, but also where they are expressed. Here we describe a detailed method for fluorescent in situ hybridization (FISH) combined with immunofluorescent labeling for cell-type markers in formalin fixed paraffin embedded (FFPE) sections along with modifications required to adapt the protocol for primary neurons grown in culture. We have combined the specificity and stability of locked nucleic acid (LNA) probes with tyramide signal amplification. To prevent loss of small RNA species, we performed post-fixation with ethylcarbodiimide (EDC). Additionally by omitting protease digestion and using only high temperature with sodium citrate buffer for FFPE sections, we were able to perform immunolabeling for proteins concurrently with in situ hybridization without compromising efficacy of either procedure.

Chaudhuri, Amrita D.; Yelamanchili, Sowmya V.; Fox, Howard S.

2013-01-01

159

Rapid prenatal diagnosis of chromosomal aneuploidies by fluorescence in situ hybridization: Clinical experience with 4,500 specimens  

SciTech Connect

Detection of chromosome aneuploidies in uncultured amniocytes is possible using fluorescence in situ hybridization (FISH). The authors herein describe the results of the first clinical program which utilized FISH for the rapid detection of chromosome aneuploidies in uncultured amniocytes. FISH was performed on physician request, as an adjunct to cytogenetics in 4,500 patients. Region-specific DNA probes to chromosomes 13, 18, 21, X, and Y were used to determine ploidy by analysis of signal number in hybridized nuclei. A sample was considered to be euploid when all autosomal probes generated two hybridization signals and when a normal sex chromosome pattern was observed in greater than or equal to 80% of hybridized nuclei. A sample was considered to be aneuploid when greater than or equal to 70% of hybridized nuclei displayed the same abnormal hybridization pattern for a specific probe. Of the attempted analyses, 90.2% met these criteria and were reported as informative to referring physicians within 2 d of receipt. Based on these reporting parameters, the overall detection rate for aneuploidies was 73.3% (107/146), with an accuracy of informative results for aneuploidies of 93.9% (107/114). Compared to cytogenetics, the accuracy of all informative FISH results, euploid and aneuploid, was 99.8%, and the specificity was 99.9%. In those pregnancies where fetal abnormalities had been observed by ultrasound, referring physicians requested FISH plus cytogenetics at a significantly higher rate than they requested cytogenetics alone. The current prenatal FISH protocol is not designed to detect all chromosome abnormalities and should only be utilized as an adjunctive test to cytogenetics. This experience demonstrates that FISH can provide a rapid and accurate clinical method for prenatal identification of chromosome aneuploidies. 40 refs., 1 fig., 5 tabs.,

Ward, B.E.; Gersen, S.L.; Carelli, M.P.; McGuire, N.M.; Dackowski, W.R.; Klinger, K.W. (Integrated Genetics, Framingham, MA (United States)); Weinstein, M. (Integrated Genetics, West Paterson, NJ (United States)); Sandlin, C. (Integrated Genetics, Long Beach, CA (United States)); Klinger, K.W. (Integrated Genetics, Miami, FL (United States))

1993-05-01

160

Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1, 1992--December 31, 1992  

SciTech Connect

Genetic mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach generated 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

Korenberg, J.R.

1993-03-04

161

Spatial distribution analyses of natural phyllosphere-colonizing bacteria on Arabidopsis thaliana revealed by fluorescence in situ hybridization.  

PubMed

Bacterial colonizers of the aerial parts of plants, or phyllosphere, have been identified on a number of different plants using cultivation-dependent and independent methods. However, the spatial distribution at the micrometer scale of different main phylogenetic lineages is not well documented and mostly based on fluorescence-tagged model strains. In this study, we developed and applied a spatial explicit approach that allowed the use of fluorescence in situ hybridization (FISH) to study bacterial phylloplane communities of environmentally grown Arabidopsis thaliana. We found on average 5.4?×?10(6) bacteria?cm(-2) leaf surface and 1.5?×?10(8) bacteria?g(-1) fresh weight. Furthermore, we found that the total biomass in the phylloplane was normally distributed. About 31% of the bacteria found in the phylloplane did not hybridize to FISH probes but exhibited infrared autofluorescence indicative for aerobic anoxygenic phototrophs. Four sets of FISH probes targeting Alphaproteobacteria, Betaproteobacteria, Actinobacteria and Bacteroidetes were sufficient to identify all other major contributors of the phylloplane community based on general bacterial probing. Spatial aggregation patterns were observed for all probe-targeted populations at distances up to 7??m, with stronger tendencies to co-aggregate for members of the same phylogenetic group. Our findings contribute to a bottom-up description of leaf surface community composition. PMID:24725362

Remus-Emsermann, Mitja N P; Lücker, Sebastian; Müller, Daniel B; Potthoff, Eva; Daims, Holger; Vorholt, Julia A

2014-07-01

162

Chromosome analysis of nuclear power plant workers using fluorescence in situ hybridization and Giemsa assay  

PubMed Central

The aim of this study was to evaluate the genotoxic effects of ionizing radiation in vivo in exposed Bulgarian nuclear power plant workers by using classical cytogenetic and molecular cytogenetic analyses of peripheral lymphocytes. Chromosome analysis using fluorescence in situ hybrydization (FISH) and Giemsa techniques was undertaken on 63 workers and 45 administrative staff controls from the Bulgarian Nuclear Power Plant. Using the Giemsa method, the frequencies of cells studied with chromosome aberrations, dicentrics plus rings and chromosome fragments in the radiation workers were significantly higher compared with the control group (P = 0.044, P = 0.014, and P = 0.033, respectively). A significant association between frequencies of dicentrics plus rings and accumulated doses was registered (P < 0.01). In the present study, a FISH cocktail of whole chromosome paints for chromosomes 1, 4 and 11 was used. A significant association between frequency of translocations and accumulated doses was also observed (P < 0.001). Within the control group, a correlation was found between age and the spontaneous frequency of translocations. No correlation was found between smoking status and frequency of translocations. When compared with the control group, workers with accumulated doses up to 100 mSv showed no increase in genome translocation frequency, whereas workers with accumulated doses from 101 to 200 mSv showed a statistically significant doubling of genome translocation frequency (P = 0.009). Thus, in cases of chronic exposure and for purposes of retrospective dosimetry, the genome frequency of translocations is a more useful marker for evaluation of genotoxic effects than dicentric frequency.

Hristova, Rositsa; Hadjidekova, Valeria; Grigorova, Mira; Nikolova, Teodora; Bulanova, Minka; Popova, Ljubomira; Staynova, Albena; Benova, Donka

2013-01-01

163

Improving the accuracy of pancreatobiliary tract cytology with fluorescence in situ hybridization: a molecular test with proven clinical success.  

PubMed

The detection of aneuploidy by fluorescence in situ hybridization (FISH) has revolutionized how laboratories diagnose cholangiocarcinoma and pancreatic adenocarcinoma using cytology specimens. Numerous clinical studies have demonstrated that FISH increases the diagnostic sensitivity of routine cytology for detecting pancreatobiliary tract malignancy with minimal decreases in clinical specificity. FISH also provides useful information in difficult clinical scenarios, including the assessment of patients with biliary strictures who have equivocal cytology results and the assessment of patients with primary sclerosing cholangitis who have clinical features suggestive of malignancy. The improved ability to detect pancreatobiliary tract cancers offers the possibility of earlier detection when patients are amenable to surgical intervention and can decrease health care costs by reducing the amount of clinical evaluation required to arrive at a cancer diagnosis. Cytopathology personnel should maintain familiarity with molecular cytology testing methodologies, because morphologic and aneuploidy assessment of tumors will continue to be an integral part of large-scale genome analyses of individual tumors. PMID:23633236

Kipp, Benjamin R; Barr Fritcher, Emily G; Pettengill, Jennifer E; Halling, Kevin C; Clayton, Amy C

2013-11-01

164

Design and application of two oligonucleotide probes for the identification of Geodermatophilaceae strains using fluorescence in situ hybridization (FISH).  

PubMed

Bacteria of the family of Geodermatophilaceae are actively involved in the decay processes [Urzě, C. and Realini, M. (1998) Int Biodeterior Biodegrad 42: 45-54; Urzě, C., Salamone, P., Schumann, P., and Stackebrandt, E. (2000) Int J Syst Evol Microbiol 50: 529-536] of stone monuments. Characterization of isolates includes phenotypic, chemotaxonomic and genetic analysis often requiring long-term procedures. The use of specific probes for members of Geodermatophilaceae family could be useful for the easy detection of those strains colonizing rock surfaces and involved in the biodeterioration. Two 16S rRNA-targeted oligonucleotide probes were designed for the specific detection of members of the family Geodermatophilaceae using fluorescence in situ hybridization (FISH); one probe specific for members of the two genera Geodermatophilus/Blastococcus and the second for members of the genus Modestobacter. PMID:15186346

Urzě, Clara; La Cono, Violetta; Stackebrandt, Erko

2004-07-01

165

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

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

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

2006-01-01

166

Fluorescence in situ hybridization mapping of esophagectomy specimens from patients with Barrett's esophagus with high-grade dysplasia or adenocarcinoma.  

PubMed

The progression of intestinal metaplasia to esophageal adenocarcinoma in patients with Barrett's esophagus is partly driven by chromosomal alterations that activate oncogenes and inactivate tumor suppressor genes. The goal of this study was to determine how alterations of 4 frequently affected genes correlate with the range of histopathologic lesions observed in resected esophagi of patients with Barrett's esophagus. Fluorescence in situ hybridization was used to assess 83 tissue sections from 10 Barrett's esophagus esophagogastrectomy specimens for chromosomal alterations of 8q24 (MYC), 9p21 (CDKN2A; alias P16), 17q12 (ERBB2), and 20q13.2 (ZNF217). Histologic lesions assessed included gastric metaplasia (n = 8), intestinal metaplasia (n = 43), low-grade dysplasia (n = 28), high-grade dysplasia (n = 25), and adenocarcinoma (n = 16). Histologic maps showing the correlation between fluorescence in situ hybridization abnormalities and corresponding histology were created for all patients. Chromosomal abnormalities included 9p21 loss, single locus gain, and polysomy. A greater number of chromosomal alterations were detected as the severity of histologic diagnosis increased from intestinal metaplasia to adenocarcinoma. All patients had alterations involving the CDKN2A gene. CDKN2A loss was the only abnormality detected in 20 (47%) of 43 areas of intestinal metaplasia. Polysomy, the most common abnormality in dysplastic epithelium and adenocarcinoma, was observed in 16 (57%) of 28 low-grade dysplasia, 22 (88%) of 25 high-grade dysplasia, and 16 (100%) of 16 adenocarcinoma. The findings of this study improve our understanding of the role that chromosomal instability and alterations of tumor suppressor genes such as CDKN2A and oncogenes such as ERBB2 play in the progression of intestinal metaplasia to adenocarcinoma in patients with Barrett's esophagus. PMID:21820152

Brankley, Shannon M; Fritcher, Emily G Barr; Smyrk, Thomas C; Keeney, Matthew E; Campion, Michael B; Voss, Jesse S; Clayton, Amy C; Wang, Kenneth K; Lutzke, Lori S; Kipp, Benjamin R; Halling, Kevin C

2012-02-01

167

Capture antibody targeted fluorescence in situ hybridization (CAT-FISH): dual labeling allows for increased specificity in complex samples.  

PubMed

Pathogen detection using biosensors is commonly limited due to the need for sensitivity and specificity in detecting targets within mixed populations. These issues were addressed through development of a dual labeling method that allows for both liquid-phase fluorescence in situ hybridization (FISH) and capture antibody targeted detection (CAT-FISH). CAT-FISH was developed using Escherichia coli O157:H7 and Staphylococcus aureus as representative bacteria, and processing techniques were evaluated with regard to FISH intensities and antibody recognition. The alternative fixative solution, methacarn, proved to be superior to standard solid-phase paraformaldehyde fixation procedures, allowing both FISH labeling and antibody recognition. CAT-FISH treated cells were successfully labeled with FISH probes, captured by immunomagnetic separation using fluorescent cytometric array beads, and detected using a cytometric array biosensor. CAT-FISH treated cells were detectable with LODs comparable to the standard antibody-based technique, (~10(3)cells/ml in PBS), and the technique was also successfully applied to two complex matrices. Although immunomagnetic capture and detection using cytometric arrays were demonstrated, CAT-FISH is readily applicable to any antibody-based fluorescence detection platform, and further optimization for sensitivity is possible via inclusion of fluorescently tagged antibodies. Since the confidence level needed for positive identification of a detected target is often paramount, CAT-FISH was developed to allow two separate levels of specificity, namely nucleic acid and protein signatures. With proper selection of FISH probes and capture antibodies, CAT-FISH may be used to provide rapid detection of target pathogens from within complex matrices with high levels of confidence. PMID:22212757

Stroot, Joyce M; Leach, Kelly M; Stroot, Peter G; Lim, Daniel V

2012-02-01

168

Cultivation-Independent, Semiautomatic Determination of Absolute Bacterial Cell Numbers in Environmental Samples by Fluorescence In Situ Hybridization  

PubMed Central

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has found widespread application for analyzing the composition of microbial communities in complex environmental samples. Although bacteria can quickly be detected by FISH, a reliable method to determine absolute numbers of FISH-stained cells in aggregates or biofilms has, to our knowledge, never been published. In this study we developed a semiautomated protocol to measure the concentration of bacteria (in cells per volume) in environmental samples by a combination of FISH, confocal laser scanning microscopy, and digital image analysis. The quantification is based on an internal standard, which is introduced by spiking the samples with known amounts of Escherichia coli cells. This method was initially tested with artificial mixtures of bacterial cultures and subsequently used to determine the concentration of ammonia-oxidizing bacteria in a municipal nitrifying activated sludge. The total number of ammonia oxidizers was found to be 9.8 × 107 ± 1.9 × 107 cells ml?1. Based on this value, the average in situ activity was calculated to be 2.3 fmol of ammonia converted to nitrite per ammonia oxidizer cell per h. This activity is within the previously determined range of activities measured with ammonia oxidizer pure cultures, demonstrating the utility of this quantification method for enumerating bacteria in samples in which cells are not homogeneously distributed.

Daims, Holger; Ramsing, Niels B.; Schleifer, Karl-Heinz; Wagner, Michael

2001-01-01

169

Physical assignment of six type I anchor loci to bovine chromosome 19 by fluorescence in situ hybridization.  

PubMed

A bovine bacterial artificial chromosome (BAC) library was screened for the presence of eight type I anchor loci previously used within hybrid somatic cells and an interspecies hybrid backcross to construct a genome map of bovine chromosome 19 (BTA19). Six out of eight loci were identified in the BAC library (NF1, CRYB1, CHRNB1, TP53, GH1 and P4HB). The BACs were then used in single-colour fluorescence in situ hybridization (FISH) to assign these genes to BTA19 band locations. Gene order was determined by single-colour FISH, and was confirmed by dual-colour FISH to mitotic and meiotic chromosomes. The order, centromere-NF1-CRYB1-CHRNB1-TP53-GH1-P4HB, was in agreement with the order determined by linkage analyses. In addition, the order of CHRNB1 and TP53, previously unresolved by linkage analyses, was established. These data provide high-resolution cytogenetic anchorage of the BTA19 genome map from chromosome bands 14-22. PMID:9699273

Gallagher, D S; Yang, Y P; Burzlaff, J D; Womack, J E; Stelly, D M; Davis, S K; Taylor, J F

1998-04-01

170

Genotype-phenotype correlation in satellited 1p chromosome: Importance of fluorescence in situ hybridization (FISH) applications  

SciTech Connect

Fluorescence in situ hybridization (FISH) was used to delineate the structural rearrangement of two satellited 1p chromosomes identified in a prenatal and in a postnatal case. Prenatal case: chromosome analysis of amniotic cells on a 37-year-old G2, PO, SAb1 woman, referred for advanced maternal age, revealed a 46,XX,1ps karyotype. Parental chromosome analyses showed that the satellited chromosome was paternal in origin. The satellited 1p did not stain with NOR and DA-DAPI. FISH using a probe specific for the rDNA, 18S and 28S genes also showed no hybridization to the satellited 1p. Using two different probes specific for 1p36.3 showed hybridization to both chromosomes 1p. This indicated that the terminal band was most likely present in the fetus and chromosomally balanced like the father. The pregnancy was continued and a phenotypically normal female was born. Postnatal case: Chromosome analysis of peripheral lymphocytes was performed on a 3 1/2-year old female with multiple congenital anomalies including growth retardation, developmental delay, upslanted palpebral fissures, thick eyebrows, esotropia, seizures, and mental retardation. She was born to a 14-year old, G1, PO mother, after a full-term pregnancy, by cesarian-section due to breech presentation. Her one-year-old brother is reportedly in good health. Chromosome analysis revealed a de novo 46,XX,lps. NOR and DA-DAPI stains were positive indicating the satellites are most likely chromosome 15 in origin. FISH using the rDNA probes also showed a hybridization to the 1ps. The two 1p36.3 probes showed only one signal to the normal chromosome 1, indicating a deletion which resulted in monosomy of 1p36.3. The clinical features of the child correlates with published cases of the terminal deletions of 1p.

Habibian, R.; Hajianpour, M.J.; Hajianpour, A.K. [Alfigen/The Genetics Institute, Pasadena, CA (United States)] [and others

1994-09-01

171

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

PubMed Central

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.

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

2014-01-01

172

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

Microsoft Academic Search

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,

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

1998-01-01

173

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

PubMed Central

We report on the rapid and specific detection of bacteria commonly isolated from clinical specimens from cystic fibrosis (CF) patients by fluorescent in situ hybridization (FISH). On the basis of comparative sequence analysis, we designed oligonucleotide probes complementary to species-specific 16S rRNA regions of these microorganisms and demonstrated the specificities of the probes by hybridization of different remotely related as well as closely related reference strains. Furthermore, in a pilot project we investigated 75 sputum samples and 10 throat swab specimens from CF patients by FISH and detected Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, Haemophilus influenzae, and Staphylococcus aureus within these specimens. The specificity of FISH was 100% in comparison to the results of conventional microbial culture. In contrast, the sensitivity of standard laboratory cultivation was moderately higher, since the limit for microscopic detection of bacteria within sputum samples by FISH was approximately 4 × 105 CFU/ml of sputum (resulting in a 90% sensitivity 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 a valuable additional method for the rapid and specific detection of bacteria in clinical samples from CF patients, in particular, patients with pulmonary exacerbations.

Hogardt, Michael; Trebesius, Karlheinz; Geiger, Anna M.; Hornef, Mathias; Rosenecker, Josef; Heesemann, Jurgen

2000-01-01

174

Processing Deep-Sea Particle-Rich Water Samples for Fluorescence In Situ Hybridization: Consideration of Storage Effects, Preservation, and Sonication  

Microsoft Academic Search

Particles are often regarded as microniches of enhanced microbial production and activities in the pelagic ocean and are vehicles of vertical material transport from the euphotic zone to the deep sea. Fluorescence in situ hybridization (FISH) can be a useful tool to study the microbial community structures associated with these particles, and thus their ecological significance, yet an appropriate protocol

Phyllis Lam; James P. Cowen

2004-01-01

175

Improved 16S rRNA-targeted probe set for analysis of sulfate-reducing bacteria by fluorescence in situ hybridization  

Microsoft Academic Search

An updated dataset of in silico specificities for 54 previously published 16S rRNA-targeted oligonucleotides was assembled to provide guidance for reliable fluorescence in situ hybridization (FISH) analysis of sulfate-reducing bacteria. Additionally, six new FISH probes were developed for major deltaproteobacterial taxa, including a probe trio targeting most Deltaproteobacteria and Gemmatimonadetes.

Sebastian Lücker; Doris Steger; Kasper Urup Kjeldsen; Barbara J. MacGregor; Michael Wagner; Alexander Loy

2007-01-01

176

Fluorescence in situ Hybridization with Human Chromosome-Specific Libraries: Detection of Trisomy 21 and Translocations of Chromosome 4  

Microsoft Academic Search

Chromosomes can be specifically stained in metaphase spreads and interphase nuclei by in situ hybridization with entire chromosome-specific DNA libraries. Unlabeled human genomic DNA is used to inhibit the hybridization of sequences in the library that bind to multiple chromosomes. The target chromosome can be made at least 20 times brighter per unit length than the others. Trisomy 21 and

D. Pinkel; J. Landegent; C. Collins; J. Fuscoe; R. Segraves; J. Lucas; J. Gray

1988-01-01

177

Single-copy gene fluorescence in situ hybridization and genome analysis: Acc-2 loci mark evolutionary chromosomal rearrangements in wheat.  

PubMed

Fluorescence in situ hybridization (FISH) is a useful tool for physical mapping of chromosomes and studying evolutionary chromosome rearrangements. Here we report a robust method for single-copy gene FISH for wheat. FISH probes were developed from cDNA of cytosolic acetyl-CoA carboxylase (ACCase) gene (Acc-2) and mapped on chromosomes of bread wheat, Triticum aestivum L. (2n?=?6x?=?42, AABBDD), and related diploid and tetraploid species. Another nine full-length (FL) cDNA FISH probes were mapped and used to identify chromosomes of wheat species. The Acc-2 probe was detected on the long arms of each of the homoeologous group 3 chromosomes (3A, 3B, and 3D), on 5DL and 4AL of bread wheat, and on homoeologous and nonhomoeologous chromosomes of other species. In the species tested, FISH detected more Acc-2 gene or pseudogene sites than previously found by PCR and Southern hybridization analyses and showed presence/absence polymorphism of Acc-2 sequences. FISH with the Acc-2 probe revealed the 4A-5A translocation, shared by several related diploid and polyploid species and inherited from an ancestral A-genome species, and the T. timopheevii-specific 4A(t)-3A(t) translocation. PMID:23052335

Danilova, Tatiana V; Friebe, Bernd; Gill, Bikram S

2012-12-01

178

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

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

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

2007-01-01

179

Fluorescence in situ hybridization mapping of human chromosome 19: Cytogenetic band location of 540 cosmids and 70 genes or DNA markers  

Microsoft Academic Search

We report here the band location of 540 cosmids mapped to chromosome 19. The cosmids were mapped by fluorescence in situ hybridization (FISH) relative to chromosomal bands produced by DAPI\\/actinomycin staining. The cosmids are distributed throughout the chromosome, with a sampling bias for the q-arm. A detailed analysis of the distribution of three different subtelomeric and 22 pericentromeric chromosome 19

B. Trask; A. Fertitta; M. Christensen; A. Bergmann; A. Copeland; P. de Jong; H. Mohrenweiser; A. Olsen; A. Carrano; K. Tynan; J. Youngblom

1993-01-01

180

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

SciTech Connect

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.

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

181

Population Dynamics of Bifidobacterium Species in Human Feces during Raffinose Administration Monitored by Fluorescence In Situ Hybridization-Flow Cytometry?  

PubMed Central

The population dynamics of bifidobacteria in human feces during raffinose administration were investigated at the species level by using fluorescence in situ hybridization (FISH) coupled with flow cytometry (FCM) analysis. Although double-staining FISH-FCM using both fluorescein isothiocyanate (FITC) and indodicarbocyanine (Cy5) as labeling dyes for fecal samples has been reported, the analysis was interfered with by strong autofluorescence at the FITC fluorescence region because of the presence of autofluorescence particles/debris in the fecal samples. We circumvented this problem by using only Cy5 fluorescent dye in the FISH-FCM analysis. Thirteen subjects received 2 g of raffinose twice a day for 4 weeks. Fecal samples were collected, and the bifidobacterial populations were monitored using the established FISH-FCM method. The results showed an increase in bifidobacteria from about 12.5% of total bacteria in the prefeeding period to about 28.7 and 37.2% after the 2-week and 4-week feeding periods, respectively. Bifidobacterium adolescentis, the Bifidobacterium catenulatum group, and Bifidobacterium longum were the major species, in that order, at the prefeeding period, and these bacteria were found to increase nearly in parallel during the raffinose administration. During the feeding periods, indigenous bifidobacterial populations became more diverse, such that minor species in human adults, such as Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium dentium, and Bifidobacterium angulatum, proliferated. Four weeks after raffinose administration was stopped, the proportion of each major bifidobacterial species, as well as that of total bifidobacteria, returned to approximately the original values for the prefeeding period, whereas that of each minor species appeared to differ considerably from its original value. To the best of our knowledge, these results provide the first clear demonstration of the population dynamics of indigenous bifidobacteria at the species level in response to raffinose administration.

Dinoto, Achmad; Marques, Tatiana M.; Sakamoto, Kanta; Fukiya, Satoru; Watanabe, Jun; Ito, Susumu; Yokota, Atsushi

2006-01-01

182

Determination of the metabolically active fraction of benthic foraminifera by means of Fluorescent in situ Hybridization (FISH)  

NASA Astrophysics Data System (ADS)

Benthic foraminifera are an important component of the marine living biota, but protocols for investigating their viability and metabolism are still extremely limited. Classical studies on benthic foraminifera have been based on direct counting under light microscopy. Typically these organisms are stained with Rose Bengal, which binds proteins and other macromolecules, but this approach does not allow discriminating between viable and recently dead organisms. The fluorescent in situ hybridization technique (FISH) represents a potentially useful approach identifying living cells with active metabolism cells. In this work, we tested for the first time the suitability of the FISH technique based on fluorescent probes targeting the 18S rRNA, to detect these live benthic protists. The protocol was applied on the genus Ammonia, on the Miliolidae group and an attempt was made also with agglutinated species (i.e., Leptohalysis scottii and Eggerella scabra). In addition microscopic analysis of the cytoplasm colour, presence of pigments and, sometimes, those of pseudopodial activity where conducted. The results of the present study indicate that FISH targeted only live and metabolically active foraminifera. These results allowed to identify as "live", cells improperly classified as "dead" by means of the classical technique (Type I error) and vice versa to identify as dead the foraminifera without rRNA, but stained using Rose Bengal (Type II error). In addition, the comparative FISH analysis of starved and actively growing cells demonstrated that individuals with active metabolism were stained more intensively than starved cells. This finding supports the hypothesis that the physiological status of cells can be directly related with the intensity of the fluorescent signal emitted by the fluorescent probe. We conclude that the use of molecular approaches could represent a key tool for acquiring crucial information on living foraminifera specimens and for investigating their ecological role in marine sediments.

Borrelli, C.; Sabbatini, A.; Luna, G. M.; Morigi, C.; Danovaro, R.; Negri, A.

2010-10-01

183

Evaluation of the environmental specificity of Fluorescence In Situ Hybridization (FISH) using Fluorescence-Activated Cell Sorting (FACS) of probe (PSE1284)-positive cells extracted from rhizosphere soil.  

PubMed

We explicitly tested for the first time the 'environmental specificity' of traditional 16S rRNA-targeted Fluorescence In Situ Hybridization (FISH) through comparison of the bacterial diversity actually targeted in the environment with the diversity that should be exactly targeted (i.e. without mismatches) according to in silico analysis. To do this, we exploited advances in modern Flow Cytometry that enabled improved detection and therefore sorting of sub-micron-sized particles and used probe PSE1284 (designed to target Pseudomonads) applied to Lolium perenne rhizosphere soil as our test system. The 6-carboxyfluorescein (6-FAM)-PSE1284-hybridized population, defined as displaying enhanced green fluorescence in Flow Cytometry, represented 3.51±1.28% of the total detected population when corrected using a nonsense (NON-EUB338) probe control. Analysis of 16S rRNA gene libraries constructed from Fluorescence Activated Cell Sorted-recovered fluorescent populations (n=3), revealed that 98.5% (Pseudomonas spp. comprised 68.7% and Burkholderia spp. 29.8%) of the total sorted population was specifically targeted as evidenced by the homology of the 16S rRNA sequences to the probe sequence. In silico evaluation of probe PSE1284 with the use of RDP-10 probeMatch justified the existence of Burkholderia spp. among the sorted cells. The lack of novelty in Pseudomonas spp. sequences uncovered was notable, probably reflecting the well-studied nature of this functionally important genus. To judge the diversity recorded within the FACS-sorted population, rarefaction and DGGE analysis were used to evaluate, respectively, the proportion of Pseudomonas diversity uncovered by the sequencing effort and the representativeness of the Nycodenz(®) method for the extraction of bacterial cells from soil. PMID:22264503

Gougoulias, Christos; Shaw, Liz J

2012-12-01

184

Triplex in-situ hybridization  

US Patent & Trademark Office Database

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.

2002-10-08

185

Triplex in-situ hybridization  

DOEpatents

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.

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

2002-01-01

186

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

PubMed Central

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.

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

1995-01-01

187

Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.  

PubMed

Here we describe a fluorescence in situ hybridization protocol that allows for the detection of two mRNA species in fresh frozen brain tissue sections. This protocol entails the simultaneous and specific hybridization of hapten-labeled riboprobes to complementary mRNAs of interest, followed by probe detection via immunohistochemical procedures and peroxidase-mediated precipitation of tyramide-linked fluorophores. In this protocol we describe riboprobes labeled with digoxigenin and biotin, though the steps can be adapted to labeling with other haptens. We have used this approach to establish the neurochemical identity of sensory-driven neurons and the co-induction of experience-regulated genes in the songbird brain. However, this procedure can be used to detect virtually any combination of two mRNA populations at single-cell resolution in the brain, and possibly other tissues. Required controls, representative results and troubleshooting of important steps of this procedure are presented. After tissue sections are obtained, the total length of the procedure is 2-3 d. PMID:18714305

Pinaud, Raphael; Mello, Claudio V; Velho, Tarciso A; Wynne, Ryan D; Tremere, Liisa A

2008-01-01

188

Efficiency of Manual Scanning in Recovering Rare Cellular Events Identified by Fluorescence In Situ Hybridization: Simulation of the Detection of Fetal Cells in Maternal Blood  

PubMed Central

Fluorescence in situ hybridization (FISH) and manual scanning is a widely used strategy for retrieving rare cellular events such as fetal cells in maternal blood. In order to determine the efficiency of these techniques in detection of rare cells, slides of XX cells with predefined numbers (1–10) of XY cells were prepared. Following FISH hybridization, the slides were scanned blindly for the presence of XY cells by different observers. The average detection efficiency was 84% (125/148). Evaluation of probe hybridization in the missed events showed that 9% (2/23) were not hybridized, 17% (4/23) were poorly hybridized, while the hybridization was adequate for the remaining 74% (17/23). In conclusion, manual scanning is a relatively efficient method to recover rare cellular events, but about 16% of the events are missed; therefore, the number of fetal cells per unit volume of maternal blood has probably been underestimated when using manual scanning.

Emad, Ahmed; Ayub, Seemi; Samassekou, Oumar; Gregoire, Marie-Chantal; Gadji, Macoura; Ntwari, Aime; Lamoureux, Josee; Hemmings, Francis; Tafas, Triantafyllos; Kilpatrick, Michael W.; Krabchi, Kada; Drouin, Regen

2012-01-01

189

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce  

PubMed Central

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55°C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (? 500 ?L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37°C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalapeńo peppers.

Bisha, Bledar; Brehm-Stecher, Byron F.

2010-01-01

190

Quantitative fluorescence in situ hybridization measurement of telomere length in skin with/without sun exposure or actinic keratosis.  

PubMed

Chromosomal and genomic instability due to telomere dysfunction is known to play an important role in carcinogenesis. To study telomere shortening in the epidermis surrounding actinic keratosis, we measured telomere lengths of basal, parabasal, and suprabasal cells in epidermis with actinic keratosis (actinic keratosis group, n = 18) and without actinic keratosis (sun-protected, n = 15, and sun-exposed, n = 13 groups) and in actinic keratosis itself as well as in dermal fibroblasts in the 3 groups, using quantitative fluorescence in situ hybridization. Among the 3 cell types, telomeres of basal cells were not always the longest, suggesting that tissue stem cells are not necessarily located among basal cells. Telomeres of basal cells in the sun-exposed group were shorter than those in the sun-protected group. Telomeres in the background of actinic keratosis and in actinic keratosis itself and those of fibroblasts in actinic keratosis were significantly shorter than those in the controls. Our findings demonstrate that sun exposure induces telomere shortening and that actinic keratosis arises from epidermis with shorter telomeres despite the absence of any histologic atypia. PMID:24411948

Ikeda, Hiroyuki; Aida, Junko; Hatamochi, Atsushi; Hamasaki, Yoichiro; Izumiyama-Shimomura, Naotaka; Nakamura, Ken-Ichi; Ishikawa, Naoshi; Poon, Steven S; Fujiwara, Mutsunori; Tomita, Ken-Ichiro; Hiraishi, Naoki; Kuroiwa, Mie; Matsuura, Masaaki; Sanada, Yukihiro; Kawano, Youichi; Arai, Tomio; Takubo, Kaiyo

2014-03-01

191

Fluorescence in situ hybridization panel for monitoring of minimal residual disease in patients with double minute chromosomes.  

PubMed

A double minute chromosome (dmin) is a small fragment of extrachromosomal DNA bearing amplified genes observed in malignancies. We investigated the incidence and characteristics of dmins in hematologic malignancies, and the quantitative changes during the treatment follow-up. Once a dmin was observed in conventional G-banding, it was characterized using fluorescence in situ hybridization (FISH) with the panel of MYC, NMYC, and MLL probes. Quantitative changes of malignant cells were measured using G-banding and FISH during the follow up. Dmins were observed in 1.23% of patients (6/489) at the initial diagnosis including 4 with MYC amplification, 1 with MLL and 1 with NMYC. All 6 had complex karyotypes and showed short overall survival (7.7 months). In follow-up specimens, FISH detected dmins in 11 cases out of which G-banding detected dmins in 9 cases. The number of dmins detected by FISH and G-banding did not correlate well. Amplification of NMYC in dmins is reported for the first time. A FISH panel composed of frequently amplified oncogenes (MYC, NMYC, and MLL) in dmins is useful for characterization of dmins. FISH is a sensitive method in detecting dmins and will be useful in monitoring of the minimal residual disease. PMID:24211232

Jeon, Yongbum; Kim, Seon Young; Kim, Miyoung; Park, Hyun-Kyung; Lee, Sang Ho; See, Cha Ja; Kwon, Jiseok; Lee, Dong Soon

2014-04-01

192

Fluorescence in-situ hybridization technique as a diagnostic and prognostic tool in oral squamous cell carcinoma  

PubMed Central

Background and Objectives: Early diagnosis and appropriate management are of prime importance for oral squamous cell carcinoma (OSCC) in the present scenario. Molecular changes in OSCC are well documented with the occurrence of a wide range of genetic damage. Identification of the genetic damage in OSCC using various diagnostic aids is mandatory, and one of the important advances in this field is cytogenetics using fluorescence in-situ hybridization (FISH). The aim of the present study is to analyze the genetic alteration in OSCC using FISH as a diagnostic aid. Materials and Methods: Peripheral blood was analyzed in 20 clinically and histopathologically proven OSCC cases and 10 healthy controls for chromosomal alteration under standardized conditions. Results: Of the 20 OSCC cases, 7 (35%) cases showed chromosomal alterations. No cases from the control group showed any chromosomal changes. Of the positive cases in OSCC, 30% cases showed increased copy number of cyclin D1 gene and 1 (5%) case showed positivity indicating extra copy of chromosome 11p11.11-q11 region. Interpretation and Conclusion: Increased genetic damage in OSCC which is a prominent feature can be identified by the use of FISH as seen from the present study. The findings suggest that FISH can be used as a diagnostic aid in the detection of genetic changes occurring in OSCC. The present study also suggests the importance of peripheral blood as a medium for assessing cytogenetic damage in OSCC.

Sunil, PM; Ramachandran, CR; Gokul, S; Jaisanghar, N

2013-01-01

193

Multi-gene fluorescence in situ hybridization to detect cell cycle gene copy number aberrations in young breast cancer patients.  

PubMed

Breast cancer is a disease of cell cycle, and the dysfunction of cell cycle checkpoints plays a vital role in the occurrence and development of breast cancer. We employed multi-gene fluorescence in situ hybridization (M-FISH) to investigate gene copy number aberrations (CNAs) of 4 genes (Rb1, CHEK2, c-Myc, CCND1) that are involved in the regulation of cell cycle, in order to analyze the impact of gene aberrations on prognosis in the young breast cancer patients. Gene copy number aberrations of these 4 genes were more frequently observed in young breast cancer patients when compared with the older group. Further, these CNAs were more frequently seen in Luminal B type, Her2 overexpression, and tiple-negative breast cancer (TNBC) type in young breast cancer patients. The variations of CCND1, Rb1, and CHEK2 were significantly correlated with poor survival in the young breast cancer patient group, while the amplification of c-Myc was not obviously correlated with poor survival in young breast cancer patients. Thus, gene copy number aberrations (CNAs) of cell cycle-regulated genes can serve as an important tool for prognosis in young breast cancer patients. PMID:24621502

Li, Chunyan; Bai, Jingchao; Hao, Xiaomeng; Zhang, Sheng; Hu, Yunhui; Zhang, Xiaobei; Yuan, Weiping; Hu, Linping; Cheng, Tao; Zetterberg, Anders; Lee, Mong-Hong; Zhang, J

2014-04-15

194

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

SciTech Connect

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.

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

2006-07-15

195

Detection of MYCN Gene Amplification in Neuroblastoma by Fluorescence In Situ Hybridization: A Pediatric Oncology Group Study1  

PubMed Central

Abstract To assess the utility of fluorescence in situ hybridization (FISH) for analysis of MYCN gene amplification in neuroblastoma, we compared this assay with Southern blot analysis using tumor specimens collected from 232 patients with presenting characteristics typical of this disease. The FISH technique identified MYCN amplification in 47 cases, compared with 39 by Southern blotting, thus increasing the total number of positive cases by 21%. The major cause of discordancy was a low fraction of tumor cells (?30% replacement) in clinical specimens, which prevented an accurate estimate of MYCN copy number by Southern blotting. With FISH, by contrast, it was possible to analyze multiple interphase nuclei of tumor cells, regardless of the proportion of normal peripheral blood, bone marrow, or stromal cells in clinical samples. Thus, FISH could be performed accurately with very small numbers of tumor cells from touch preparations of needle biopsies. Moreover, this procedure allowed us to discern the heterogeneous pattern of MYCN amplification that is characteristic of neuroblastoma. We conclude that FISH improves the detection of MYCN gene amplification in childhood neuroblastomas in a clinical setting, thus facilitating therapeutic decisions based on the presence or absence of this prognostically important biologic marker.

Mathew, Prasad; Valentine, Marcus B; Bowman, Laura C; Rowe, Susan T; Nash, Michael B; Valentine, Virginia A; Cohn, Susan L; Castleberry, Robert P; Brodeur, Garrett M; Look, A Thomas

2001-01-01

196

Chromosome instability in ICF syndrome: Formation of micronuclei from multibranched chromosomes 1 demonstrated by fluorescence in situ hybridization  

SciTech Connect

We report on a new patient with immunodeficiency, centromeric heterochromatin instability, and facial anomalies (the ICF syndrome). Studies with traditional cytogenetic methods demonstrate that aberrations in this syndrome primarily involve the centromeric regions of chromosomes 1 and 16. We applied fluorescence in situ hybridization (FISH) using {open_quotes}painting{close_quotes} probes for chromosomes 1 and 16 to document the progression of centromeric instability from simple decondensation aberrations to the subsequent formation of complex multibranched chromosomes 1, and finally to the interphase aberrations of nuclear projections and micronuclei involving both chromosomes 1 and 16. The loss of the large multibranched chromosome 1 configurations from the cells as micronuclei suggests that the centromeric aberrations subsequently interfere with normal chromosome movement at anaphase in ICF syndrome. Circular areas of counterstained chromatin were observed by FISH in the micronuclei corresponding to the intertwined segments of centromeric heterochromatin seen involving multibranched chromosomes 1 in the patient`s G-banded chromosome study. The current hypothesis of recessive inheritance for this disorder suggests that the chromosomal aberrations are not a causative event in this syndrome; however, the chromosome aberrations are clearly an important basic diagnostic criterion. 22 refs., 3 figs.

Sawyer, J.R.; Swanson, C.M.; Wheeler, G. [Univ. of Arkansas, Little Rock, AR (United States)] [and others

1995-03-27

197

Comprehensive Analysis of ETS Family Members in Melanoma by Fluorescence In Situ Hybridization Reveals Recurrent ETV1 Amplification.  

PubMed

E26 transformation-specific (ETS) transcription factors are known to be involved in gene aberrations in various malignancies including prostate cancer; however, their role in melanoma oncogenesis has yet to be fully explored. We have completed a comprehensive fluorescence in situ hybridization (FISH)-based screen for all 27 members of the ETS transcription factor family on two melanoma tissue microarrays, representing 223 melanomas, 10 nevi, and 5 normal skin tissues. None of the melanoma cases demonstrated ETS fusions; however, 6 of 114 (5.3%) melanomas were amplified for ETV1 using a break-apart FISH probe. For the six positive cases, locus-controlled FISH probes revealed that two of six cases were amplified for the ETV1 region, whereas four cases showed copy gains of the entire chromosome 7. The remaining 26 ETS family members showed no chromosomal aberrations by FISH. Quantitative polymerase chain reaction showed an average 3.4-fold (P value = .00218) increased expression of ETV1 in melanomas, including the FISH ETV1-amplified cases, when compared to other malignancies (prostate, breast, and bladder carcinomas). These data suggest that a subset of melanomas overexpresses ETV1 and amplification of ETV1 may be one mechanism for achieving high gene expression. PMID:23908683

Mehra, Rohit; Dhanasekaran, Saravana M; Palanisamy, Nallasivam; Vats, Pankaj; Cao, Xuhong; Kim, Jung H; Kim, David Sl; Johnson, Timothy; Fullen, Douglas R; Chinnaiyan, Arul M

2013-08-01

198

Infertility in a new 46, XX male with positive SRY confirmed by fluorescence in situ hybridization: a case report.  

PubMed

The 46, XX male syndrome (de la Chapelle syndrome or 46, XX testicular disorder of sex development) is a rare form of sex reversal with complex mechanisms leading to a large spectrum of clinical manifestations ranging from ambiguous genitalia in the newborn to normal male phenotype. Therefore, diagnosis is established either pre- or early postnatal, or in adult life due to male infertility. In some cases, subtle clinical signs during childhood and puberty may be overlooked. A 28-year-old married man presented with azoospermia without erectile dysfunction. Between 9-14 years he was examined for the small testes and under-masculinized external genitalia but the diagnosis was not further clarified. At presentation, hormonal laboratory evaluation revealed hypergonadotropic hypogonadism. Chromosome analysis showed a 46, XX karyotype and translocation of SRY (testis-determining factor) from chromosome Y to chromosome X was identified by fluorescence in situ hybridization (FISH). Despite early subtle clinical signs of abnormal sexual development in this new 46, XX male syndrome, medical investigations were triggered by infertility. PMID:19205451

Pepene, C E; Coman, I; Mihu, D; Militaru, M; Duncea, I

2008-01-01

199

Fluorescence in situ hybridization (FISH) using non-commercial probes in the diagnosis of clinically suspected microdeletion syndromes  

PubMed Central

Background & objectives: Microdeletion syndromes are characterized by small (<5 Mb) chromosomal deletions in which one or more genes are involved. These are frequently associated with multiple congenital anomalies. The phenotype is the result of haploinsufficiency of genes in the critical interval. Fluorescence in situ hybridization (FISH) technique is commonly used for precise genetic diagnosis of microdeletion syndromes. This study was conducted to assess the role of FISH in the diagnosis of suspected microdeletion syndrome. Methods: FISH was carried out on 301 clinically suspected microdeletion syndrome cases for the confirmation of clinical diagnosis using non-commercial probes. Of these, 177 cases were referred for 22q11.2 microdeletion, 42 cases were referred for William syndrome, 38 cases were referred for Prader Willi/Angelman and 44 cases were referred for other suspected microdeletion syndromes. Results: FISH was confirmatory in 23 cases only (7.6%). There were 17 cases of 22q11.2 microdeletion, four cases of Prader Willi syndrome and two cases of William syndrome. Interpretation & conclusion: We conclude that FISH should not be the method of choice for clinically suspected microdeletion syndromes. We propose to follow strict clinical criteria for FISH testing or preferably to follow better methods (genotype first approach). Whole genome screening may be used as first line of test and FISH may be used for confirmation of screening result, screening of family members and prenatal diagnosis.

Halder, Ashutosh; Jain, Manish; Chaudhary, Isha; Gupta, Neerja; Kabra, Madhulika

2013-01-01

200

Grazing of a Tetrahymena sp. on Adhered Bacteria in Percolated Columns Monitored by In Situ Hybridization with Fluorescent Oligonucleotide Probes  

PubMed Central

Predation of attached Pseudomonas putida mt2 by the small ciliate Tetrahymena sp. was investigated with a percolated column system. Grazing rates were examined under static and dynamic conditions and were compared to grazing rates in batch systems containing suspended prey. The prey densities were 2 × 108 bacteria per ml of pore space and 2 × 108 bacteria per ml of suspension, respectively. Postingestion in situ hybridization of bacteria with fluorescent oligonucleotide probes was used to quantify ingestion. During 30 min, a grazing rate of 1,382 ± 1,029 bacteria individual?1 h?1 was obtained with suspended prey; this was twice the grazing rate observed with attached bacteria under static conditions. Continuous percolation at a flow rate of 73 cm h?1 further decreased the grazing rate to about 25% of the grazing rate observed with suspended prey. A considerable proportion of the protozoans fed on neither suspended bacteria nor attached bacteria. The transport of ciliates through the columns was monitored at the same time that predation was monitored. Less than 20% of the protozoans passed through the columns without being retained. Most of these organisms ingested no bacteria, whereas the retained protozoans grazed more efficiently. Retardation of ciliate transport was greater in columns containing attached bacteria than in bacterium-free columns. We propose that the correlation between grazing activity and retardation of transport is a consequence of the interaction between active predators and attached bacteria.

Eisenmann, Heinrich; Harms, Hauke; Meckenstock, Rainer; Meyer, Elisabeth I.; Zehnder, Alexander J. B.

1998-01-01

201

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

PubMed Central

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.

Strapoc, Dariusz; Huizinga, Brad; Lidstrom, Ulrika; Ashby, Matt; Macalady, Jennifer L.

2012-01-01

202

Analysis of methanogenic activity in a thermophilic-dry anaerobic reactor: Use of fluorescent in situ hybridization  

SciTech Connect

Methanogenic activity in a thermophilic-dry anaerobic reactor was determined by comparing the amount of methane generated for each of the organic loading rates with the size of the total and specific methanogenic population, as determined by fluorescent in situ hybridization. A high correlation was evident between the total methanogenic activity and retention time [-0.6988Ln(x) + 2.667] (R{sup 2} 0.8866). The total methanogenic activity increased from 0.04 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} to 0.38 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} while the retention time decreased, augmenting the organic loading rates. The specific methanogenic activities of H{sub 2}-utilizing methanogens and acetate-utilizing methanogens increased until they stabilised at 0.64 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1} and 0.33 x 10{sup -8} mLCH{sub 4} cell{sup -1}day{sup -1}, respectively. The methanogenic activity of H{sub 2}-utilizing methanogens was higher than acetate-utilizing methanogens, indicating that maintaining a low partial pressure of hydrogen does not inhibit the acetoclastic methanogenesis or the anaerobic process.

Montero, B. [Department of Chemical Engineering, Food and Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cadiz, Campus Rio San Pedro s/n, 11510-Puerto Real, Cadiz (Spain)], E-mail: blanca.montero@uca.es; Garcia-Morales, J.L.; Sales, D.; Solera, R. [Department of Chemical Engineering, Food and Environmental Technologies, Faculty of Marine and Environmental Sciences, University of Cadiz, Campus Rio San Pedro s/n, 11510-Puerto Real, Cadiz (Spain)

2009-03-15

203

A new complex variant Philadelphia chromosome, t(1;9;22)ins(17;22), characterized by fluorescence in situ hybridization in an adult ALL  

Microsoft Academic Search

A new complex variant Philadelphia chromosome was detected in a 65-year-old man with acute, pre-B, lymphoblastic leukemia (ALL). The classic cytogenetic analysis identified an apparently balanced three-way translocation t(1;9;22)(q25;q34;q11.2). Fluorescence in situ hybridization (FISH) studies confirmed the translocation and showed bcr\\/abl fusion on the der(22). However, these studies revealed that the distal part of the bcr gene was not translocated

Yao-Shan Fan; Kamilia Rizkalla; Robert M Barr

1999-01-01

204

High-Temperature Fluorescent In Situ Hybridization for Detecting Escherichia coli in Seawater Samples, Using rRNA-Targeted Oligonucleotide Probes and Flow Cytometry  

PubMed Central

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E.coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with a hybridization time of less than 30 min, with performance equivalent to or better than the standard protocol in terms of the fluorescence signal intensity and the FISH hybridization efficiency (i.e., the percentage of hybridized cells giving satisfactory fluorescence intensity): (i) one-step FISH (hybridization is conducted at 60 to 75°C for 30 min) and (ii) two-step FISH (pretreatment in a 90°C water bath for 5 min and a hybridizing step at 50 to 55°C for 15 to 20 min). We also found that satisfactory fluorescence signal intensity does not necessarily guarantee satisfactory hybridization efficiency and the tightness of the targeted population when analyzed with a flow cytometer. We subsequently successfully applied the optimized protocols to E. coli-spiked seawater samples, i.e., obtained flow cytometric signatures where the E. coli population was well separated from other particles carrying fluorescence from nonspecific binding to probes or from autofluorescence, and had a good recovery rate of the spiked E. coli cells (90%).

Tang, Ying Zhong; Gin, Karina Yew Hoong; Lim, Tok Hoon

2005-01-01

205

Diagnostic Utility of Multiprobe Fluorescence in situ Hybridization Assay for Detecting Cytogenetic Aberrations in Acute Leukemia  

PubMed Central

Background Specific cytogenetic aberrations detected by conventional karyotyping or FISH play a major role in the diagnosis, prognosis, and treatment of patients with acute leukemia. The FISH technique enhances the capacity of conventional karyotyping to detect subtle chromosomal aberrations. Multiprobe FISH assay (Cytocell, UK) can hybridize multiple probes to a single slide, thereby increasing the detection rate of cytogenetic aberrations. This study aimed to evaluate multiprobe FISH in detecting cytogenetic abnormalities in acute leukemia. Methods Thirty newly diagnosed acute leukemia patients who attended the hematology clinic at Dong-A University Hospital from October 2008 to October 2012 were enrolled in the study. The multiprobe FISH results were compared with those of G-banding. Results Multiprobe FISH detected the chromosomal aberrations identified by G-banding, as well as additional aberrations in 6 of 30 (20.0%) cases, which included ETV6/RUNX1 translocation, p16 deletion, TP53 deletion, and IGH break-apart. Conclusions The multiprobe FISH assay was a more sensitive and reliable technique compared with G-banding. It was also more cost-effective and yielded faster results.

Kim, Bo-Ram; Choi, Jae-Lim; Kim, Ji-Eun; Woo, Kwang-Sook; Kim, Kyeong-Hee; Kim, Jeong-Man; Kim, Sung-Hyun

2014-01-01

206

Cytogenetic, fluorescence in situ hybridization, and molecular characterization of chronic myeloid leukemia in chronic phase with four BCR/ABL1 fusion signals: a case report.  

PubMed

We report a case of chronic myeloid leukemia in chronic stage with 48 chromosomes and four BCR/ABL1 fusion signals on two out of three chromosomes 9 and two signals on the two Philadelphia chromosomes. These abnormalities were detected by both conventional cytogenetic analysis and metaphase and interphase fluorescence in situ hybridization studies in approximately 90% of the cells at diagnosis. Real-time-polymerase chain reaction studies on peripheral blood showed b3a2(p210) and e1a2(p190) BCR/ABL1 fusion transcripts. During treatment with imatinib, the patient was asymptomatic with hematological remission. Cytogenetic and fluorescence in situ hybridization analysis revealed that only 6.6% of cells had the initial majority line karyotype, with disappearance of the p210 but increased p190 transcript, which led to the treatment being changed. We discuss the implication of cytogenetic and molecular alterations in the patient's evolution and treatment. PMID:19837272

Vargas, Maria Teresa; Portero, Maria Angeles; Rodríguez, Alicia; Reyes, Juana; Fernández-Novoa, Carmen

2009-11-01

207

Potential Errors with Rapid Analysis Techniques: Partial Duplication 21q Resulting from a Paternal Paracentric Insertion Uncovered in Chorionic Villus Sampling by Fluorescence in situ Hybridization  

Microsoft Academic Search

We report on partial duplication 21q resulting from a paternal insertion identified during prenatal diagnosis. While performing interphase fluorescence in situ hybridization (I-FISH), we were able to identify 3 signals of the LSI 21 Spectrum Orange probe with chorionic villus sampling. Using standard cytogenetic analysis, I-FISH and GTG banding, structural aberrations in 21q in the parents and in the fetus

N. Ehrhardt; A. Kujat; R. Faber; L.-C. Horn; U. G. Froster

2009-01-01

208

Fluorescence in situ hybridization analysis of minimal residual disease and the relevance of the der(9) deletion in imatinib-treated patients with chronic myeloid leukemia.  

PubMed

Forty-six patients with chronic myeloid leukemia receiving imatinib mesylate (39 in chronic phase, one in accelerated phase, and six in blastic crisis), were studied for a 20-62 month follow-up period by cytogenetics and fluorescence in situ hybridization using dual-color, dual-fusion BCR and ABL probes. This approach provided valuable results for disease management of analysis. PMID:16818290

Calabrese, Giuseppe; Fantasia, Donatella; Di Gianfilippo, Rita; Stuppia, Liborio; Di Lorenzo, Roberto; Palka, Giandomenico

2006-07-01

209

Concurrent use of flow cytometry and fluorescence in-situ hybridization techniques for detecting faulty meiosis in a human sperm sample  

Microsoft Academic Search

5To whom correspondence should be addressed Routine semen analysis in an infertile patient revealed severe teratospermia associated with malformation of head and tail in 100% of the sperm cells. Flow cytometry and fluorescence in-situ hybridization (FISH) were shown to supplement routine semen analysis by providing information on the sperm chromatin. Using flow cytometry, propidium iodide-stained spermatozoa from the same sperm

R. Weissenberg; A. Aviram; R. Golan; L. M. Lewin; J. Levron; I. Madgar; J. Dor; G. Barkai; B. Goldman

1998-01-01

210

Identification of dominant microbial community in aerophilic biofilm reactors by fluorescence in situ hybridization and PCR-denaturing gradient gel electrophoresis  

Microsoft Academic Search

This study was conducted by combining fluorescence in situ hybridization (FISH) performed on 16S rRNA and polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE)\\u000a with 16S CTOs primers to characterize the nitrifying microbial communities in biofilm processes, which were tested to retrofit\\u000a the S municipal wastewater treatment plant in Busan, Korea. Four aerophilic biofilm reactors were operated with hydraulic\\u000a retention

Youngo Kim; Taeho Lee; Sanghyup Lee

2009-01-01

211

Single-Gene Detection and Karyotyping Using Small-Target Fluorescence in Situ Hybridization on Maize Somatic Chromosomes  

PubMed Central

Combined with a system for identifying each of the chromosomes in a genome, visualizing the location of individual genetic loci by fluorescence in situ hybridization (FISH) would aid in assembling physical and genetic maps. Previously, large genomic clones have been successfully used as FISH probes onto somatic chromosomes but this approach is complicated in species with abundant repetitive elements. In this study, repeat-free portions of sequences that were anchored to particular chromosomes including genes, gene clusters, large cDNAs, and portions of BACs obtained from public databases were used to label the corresponding physical location using FISH. A collection of probes that includes at least one marker on each chromosome in the maize complement was assembled, allowing a small-target karyotyping system to be developed. This set provides the foundation onto which additional loci could be added to strengthen further the ability to perform chromosomal identification in maize and its relatives. The probes were demonstrated to produce signals in several wild relatives of maize, including Zea luxurians, Z. diploperennis, and Tripsacum dactyloides.

Lamb, Jonathan C.; Danilova, Tatiana; Bauer, Matthew J.; Meyer, Julie M.; Holland, Jennifer J.; Jensen, Michael D.; Birchler, James A.

2007-01-01

212

Human cDNA mapping using fluorescence in situ hybridization. Progress report, April 1--December 31, 1992  

SciTech Connect

The ultimate goal of this proposal is to create a cDNA map of the human genome. Mapping is approached using the techniques of high resolution fluorescence in situ hybridization (FISH). This technology and the results of its application are designed to rapidly generate whole genome as tool box of expressed sequence to speed the identification of human disease genes. The results of this study are intended to dovetail with and to link the results of existing technologies for creating backbone YAC and genetic maps. In the first eight months, this approach will generate 60--80% of the expressed sequence map, the remainder expected to be derived through more long-term, labor-intensive, regional chromosomal gene searches or sequencing. The laboratory has made significant progress in the set-up phase, in mapping fetal and adult brain and other cDNAs, in testing a model system for directly linking genetic and physical maps using FISH with small fragments, in setting up a database, and in establishing the validity and throughput of the system.

Korenberg, J.R.

1993-12-31

213

Rapid identification of Acinetobacter spp. by fluorescence in situ hybridization (FISH) from colony and blood culture material  

PubMed Central

Multi-drug-resistant strains of the Acinetobacter baumannii complex cause nosocomial infections. Rapid identification of Acinetobacter spp. is desirable in order to facilitate therapeutic or hygiene decisions. We evaluated a newly designed DNA probe that can be used under standard conditions in both a microwave oven and a slide chamber for the rapid identification of Acinetobacter spp. by fluorescence in situ hybridization (FISH). Using FISH, the new probe correctly identified 81/81 Acinetobacter spp. isolates and excluded 109/109 tested non-target organisms from agar culture. Furthermore, the new probe correctly identified 7/7 Acinetobacter spp. in 214 blood cultures determined to contain Gram-negative bacteria by Gram staining. Using either the microwave oven or slide chamber technique, the new probe was able to identify Acinetobacter spp. in 100% of the samples tested. FISH used in conjunction with our newly designed probe provides an easy, cheap, precise, and rapid method for the preliminary identification of Acinetobacter spp., especially in laboratories where more sophisticated methods like matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) are not available.

Essig, A.; Hagen, R. M.; Riecker, M.; Jerke, K.; Ellison, D.; Poppert, S.

2011-01-01

214

Variant Three-Way Translocation of Inversion 16 in AML-M4Eo Confirmed by Fluorescence In Situ Hybridization Analysis  

Microsoft Academic Search

The inv(16) and t(16;16) characterize a subgroup of acute myelomonocytic leukemia (AML) with distinct morphological features and a favorable prognosis. Both cytogenetic abnormalities result in a fusion of CBF? at 16q22 and MYH11 gene at 16p13, whose detection by PCR and fluorescence in situ hybridization (FISH) is useful for diagnosis and monitoring of the disease. Variant translocations of inv(16)\\/t(16;16) are

Jose A Martinez-Climent; Ana M Comes; Esperanza Vizcarra; Shalini Reshmi; Isabel Benet; Isabel Marugan; Mar Tormo; M. Jose Terol; Carlos Solano; Cristina Arbona; Felipe Prosper; Eva Barragan; Pascual Bolufer; Janet D Rowley; Javier Garc??a-Conde

1999-01-01

215

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

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

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

216

Ecophysiological Interaction between Nitrifying Bacteria and Heterotrophic Bacteria in Autotrophic Nitrifying Biofilms as Determined by Microautoradiography-Fluorescence In Situ Hybridization  

Microsoft Academic Search

Ecophysiological interactions between the community members (i.e., nitrifiers and heterotrophic bacteria) in a carbon-limited autotrophic nitrifying biofilm fed only NH4 as an energy source were investigated by using a full-cycle 16S rRNA approach followed by microautoradiography (MAR)-fluorescence in situ hybridization (FISH). Phylogenetic differentiation (identification) of heterotrophic bacteria was performed by 16S rRNA gene sequence analysis, and FISH probes were designed

Tomonori Kindaichi; Tsukasa Ito; Satoshi Okabe

2004-01-01

217

Detection of EWS-FLI-1 fusion in Ewing's sarcoma\\/peripheral primitive neuroectodermal tumor by fluorescence in situ hybridization using formalin-fixed paraffin-embedded tissue  

Microsoft Academic Search

The balanced translocation t(11;22)(q24;q12) is specific for the Ewing's sarcoma\\/peripheral primitive neuroectodermal tumors (ESPNETs) and results in the EWSFLI-1 fusion transcript, which can be detected by reverse transcription polymerase chain reaction (RT-PCR). Recent studies also have used fluorescence in situ hybridization (FISH) to show the translocation; however, most of these have been performed on cell lines or touch preparations and

Shimareet Kumar; Svetlana Pack; Dhruv Kumar; Robert Walker; Martha Quezado; Zhengping Zhuang; Paul Meltzer; Maria Tsokos

1999-01-01

218

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

Microsoft Academic Search

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 fluoro- chrome Cy3 or horseradish peroxidase-modified oligonucleotide probes. For both basins, significant differ- ences in total prokaryotic abundance and phylogenetic composition were observed among oxic, anoxic, and transitional (redoxcline) waters. Epsilon-proteobacteria,

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

2006-01-01

219

Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA  

Microsoft Academic Search

A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization\\u000a and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of\\u000a recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences

Cindy H. Wu; Yu-Chen Hwang; Wonkyu Lee; Ashok Mulchandani; Thomas K. Wood; Marylynn V. Yates; Wilfred Chen

2008-01-01

220

Discriminating Multi-Species Populations in Biofilms with Peptide Nucleic Acid Fluorescence In Situ Hybridization (PNA FISH)  

PubMed Central

Background Our current understanding of biofilms indicates that these structures are typically composed of many different microbial species. However, the lack of reliable techniques for the discrimination of each population has meant that studies focusing on multi-species biofilms are scarce and typically generate qualitative rather than quantitative data. Methodology/Principal Findings We employ peptide nucleic acid fluorescence in situ hybridization (PNA FISH) methods to quantify and visualize mixed biofilm populations. As a case study, we present the characterization of Salmonella enterica/Listeria monocytogenes/Escherichia coli single, dual and tri-species biofilms in seven different support materials. Ex-situ, we were able to monitor quantitatively the populations of ?56 mixed species biofilms up to 48 h, regardless of the support material. In situ, a correct quantification remained more elusive, but a qualitative understanding of biofilm structure and composition is clearly possible by confocal laser scanning microscopy (CLSM) at least up to 192 h. Combining the data obtained from PNA FISH/CLSM with data from other established techniques and from calculated microbial parameters, we were able to develop a model for this tri-species biofilm. The higher growth rate and exopolymer production ability of E. coli probably led this microorganism to outcompete the other two [average cell numbers (cells/cm2) for 48 h biofilm: E. coli 2,1×108 (±2,4×107); L. monocytogenes 6,8×107 (±9,4×106); and S. enterica 1,4×106 (±4,1×105)]. This overgrowth was confirmed by CSLM, with two well-defined layers being easily identified: the top one with E. coli, and the bottom one with mixed regions of L. monocytogenes and S. enterica. Significance While PNA FISH has been described previously for the qualitative study of biofilm populations, the present investigation demonstrates that it can also be used for the accurate quantification and spatial distribution of species in polymicrobial communities. Thus, it facilitates the understanding of interspecies interactions and how these are affected by changes in the surrounding environment.

Almeida, Carina; Azevedo, Nuno F.; Santos, Silvio; Keevil, Charles W.; Vieira, Maria J.

2011-01-01

221

Community Composition of Marine Bacterioplankton Determined by 16S rRNA Gene Clone Libraries and Fluorescence In Situ Hybridization  

PubMed Central

We determined the compositions of bacterioplankton communities in surface waters of coastal California using clone libraries of 16S rRNA genes and fluorescence in situ hybridization (FISH) in order to compare the community structures inferred from these two culture-independent approaches. The compositions of two clone libraries were quite similar to those of clone libraries of marine bacterioplankton examined by previous studies. Clones from ?-proteobacteria comprised ca. 28% of the libraries, while approximately 55% of the clones came from ?-proteobacteria, which dominated the clone libraries. The Cytophaga-Flavobacter group and three others each comprised 10% or fewer of the clone libraries. The community composition determined by FISH differed substantially from the composition implied by the clone libraries. The Cytophaga-Flavobacter group dominated 8 of the 11 communities assayed by FISH, including the two communities assayed using clone libraries. On average only 10% of DAPI (4?,6?-diamidino-2-phenylindole)-stained bacteria were detected by FISH with a probe for ?-proteobacteria, but 30% of DAPI-stained bacteria appeared to be in the Cytophaga-Flavobacter group as determined by FISH. ?-Proteobacteria were greatly overrepresented in clone libraries compared to their relative abundance determined by FISH, while the Cytophaga-Flavobacter group was underrepresented in clone libraries. Our data show that the Cytophaga-Flavobacter group can be a numerically dominant component of coastal marine bacterioplankton communities.

Cottrell, Matthew T.; Kirchman, David L.

2000-01-01

222

Fluorescent in situ hybridization analysis of open lactic acid fermentation of kitchen refuse using rRNA-targeted oligonucleotide probes.  

PubMed

Reproducible amounts of lactic acid accumulate in minced kitchen refuse under open conditions with intermittent pH neutralization [Sakai et al., Food Sci. Technol. Res., 6, 140 (2000)]. Here, we showed that such pH-controlled open fermentation of kitchen refuse reproducibly resulted a selective proliferation of a major lactic acid bacterial (LAB) species. In one experiment, the predominant microorganisms isolated during the early phase (6 h) were Gammaproteobacteria. In contrast, those that predominated during the late phase (48 h) were always Lactobacillus plantarum in three independent experiments. To further quantify the microbial community within open lactic acid fermentation, we performed fluorescent in situ hybridization (FISH) analysis targeting 16S (23S) rRNA. We designed two new group-specific DNA probes: LAC722(L) was active for most LAB including the genera Lactobacillus, Pediococcus, Leuconostoc and Weisella, whereas Lplan477 was specific for L. plantarum and its related species. We then optimized sample preparation using lysozyme and hybridization conditions including temperature, as well as the formamide concentration and the salt concentration in the washing buffer. We succeeded in quantification of microorganisms in semi-solid, complex biological materials such as minced kitchen refuse by taking color microphotographs in modified RGB balance on pre-coated slides. FISH analysis of the fermentation of kitchen refuse indicated that control of the pH swing leads to domination by the LAB population in minced kitchen refuse under open conditions. We also confirmed that L. plantarum, which generates lactic acid in high quantities but with low optical activity, became the dominant microorganism in kitchen refuse during the late phase of open fermentation. PMID:16233665

Sakai, Kenji; Mori, Masatsugu; Fujii, Akira; Iwami, Yuko; Chukeatirote, Ekachai; Shirai, Yoshihito

2004-01-01

223

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

NASA Astrophysics Data System (ADS)

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.

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

2012-03-01

224

Characterization of an unbalanced de novo rearrangement by microsatellite polymorphism typing and by fluorescent in situ hybridization  

SciTech Connect

Unbalanced de novo rearrangements, difficult to characterize by conventional cytogenetic techniques, may be elucidated by molecular approaches. By dinucleotide repeat polymorphism typing and fluorescence in situ hybridization (FISH), we have defined the composition of an unbalanced de novo translocation (46,XX,15p+) in a child with multiple congenital anomalies. Use of a microsatellite repeat D5S208 (localized to 5p15) and polymerase chain reaction (PCR) analysis confirmed that the extra segment originated from the short arm of chromosome 5. Amplification of the patient`s DNA with primers for dinucleotide repeats D5S350 and D5S118 showed that the entire 5p (from 5pter to 5q11) was present in 3 copies. FISH confirmed the trisomic status of 5p, and further revealed the presence of centromeres of both chromosomes 5 and 15 on the rearranged chromosome thus delineating its dicentric nature. This information allowed us to redefine the de novo rearrangement in this patient as 46,XX,dic der(15)t(5;15)(q11;p11). 19 refs., 4 figs., 2 tabs.

Zhao, J.; Gordon, P.L.; Wilroy, R.S. Jr. [Univ. of Tennessee, Memphis, TN (United States)] [and others

1995-05-08

225

Evaluation of fluorescence in situ hybridization to detect encapsulated Bacillus pumilus SAFR-032 spores released from poly(methylmethacrylate).  

PubMed

Bacillus pumilus SAFR-032 spores originally isolated from the Jet Propulsion Laboratory spacecraft assembly facility clean room are extremely resistant to UV radiation, H(2)O(2), desiccation, chemical disinfection and starvation compared to spores of other Bacillus species. The resistance of B. pumilus SAFR-032 spores to standard industrial clean room sterilization practices is not only a major concern for medical, pharmaceutical and food industries, but also a threat to the extraterrestrial environment during search for life via spacecraft. The objective of the present study was to investigate the potential of Alexa-FISH (fluorescence in situ hybridization with Alexa Fluor® 488 labeled oligonucleotide) method as a molecular diagnostic tool for enumeration of multiple sterilant-resistant B. pumilus SAFR-032 spores artificially encapsulated in, and released via organic solvent from, a model polymeric material: poly(methylmethacrylate) (Lucite, Plexiglas). Plexiglas is used extensively in various aerospace applications and in medical, pharmaceutical and food industries. Alexa-FISH signals were not detected from spores via standard methods for vegetative bacterial cells. Optimization of a spore permeabilization protocol capitalizing on the synergistic action of proteinase-K, lysozyme, mutanolysin and Triton X-100 facilitated efficient spore detection by Alexa-FISH microscopy. Neither of the Alexa-probes tested gave rise to considerable levels of Lucite- or solvent-associated background autofluorescence, demonstrating the immense potential of Alexa-FISH for rapid quantification of encapsulated B. pumilus SAFR-032 spores released from poly(methylmethacrylate). PMID:22145981

Mohapatra, Bidyut R; La Duc, Myron T

2012-01-01

226

Community analysis of bacterial biofilms in a simulated recirculating cooling-water system by fluorescent in situ hybridization with rRNA-targeted oligonucleotide probes  

Microsoft Academic Search

An open recirculating cooling-water system feeding a modified Robbin’s Device (MRD) with synthetic cooling water to simulate the environment of a recirculating industrial cooling-water system was set up. Both mild steel and Nylon® coupons were inserted to sample biofilms at regular intervals. The community structure was determined by in situ hybridization using fluorescently labelled 16S and 23S rRNA-targeted oligonucleotide probes

R MacDonald; V. S Brözel

2000-01-01

227

Characterization of a de novo unbalanced translocation t(14q18q) using microdissection and fluorescence in situ hybridization.  

PubMed

We report on a patient with a de novo translocation between the long arms of chromosomes 14 and 18. The translocation was studied using microdissection in combination with fluorescence in situ hybridization (micro-FISH). Five copies of the chromosomes involved in the translocation were isolated by microdissection and amplified by means of degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR). Reverse chromosome painting with the biotin-labeled PCR product showed that part of the q-arm of chromosome 18 had no signal. The deletion was characterized further by FISH with band-specific probes and it was concluded that the rearrangement was unbalanced: 46,XY,t(14;18)(14pter-->14q22::18q21.1-->18qter) (18pter-->18q12.2::14q22-->14qter). The patient, who presented with psychomotor retardation, mild obesity, pes equinovarus, strabismus, and facial anomalies, is compared with previously reported patients with an interstitial deletion of band 18q12. PMID:9482648

Engelen, J J; Loots, W J; Albrechts, J C; Plomp, A S; van der Meer, S B; Vles, J S; Hamers, G J; Geraedts, J P

1998-02-01

228

Application of fluorescence in situ hybridization for the study and characterization of nitrifying bacteria in nitrifying/denitrifying wastewater treatment plants.  

PubMed

The aim of this study was to verify fluorescence in situ hybridization for the detection of nitrifying bacteria in activated sludge and biofilms, and to determine the distribution of nitrifiers in selected wastewater treatment plants (WWTPs). Both Czech and foreign WWTPs with intensification of nitrification (for example, in situ bioaugmentation of nitrification or biofilms) and without intensification were studied. The two-dimensional and three-dimensional analyses of microscopic images were focused on quantifying the parameters and their differences with regard to the arrangement, capacity and sludge age of the WWTPs. This is the first time such a study has been performed in the Czech Republic. PMID:24350498

Benakova, A; Wanner, J

2013-01-01

229

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2010 CFR

...automated FISH enumeration system is a device that consists...microscope, image analysis system, and customized software...formalin-fixed, paraffin-embedded human tissue specimens. ...Hybridization (FISH) Enumeration Systems.â See § 866.1(e)...

2010-04-01

230

21 CFR 866.4700 - Automated fluorescence in situ hybridization (FISH) enumeration systems.  

Code of Federal Regulations, 2010 CFR

...automated FISH enumeration system is a device that consists...microscope, image analysis system, and customized software...formalin-fixed, paraffin-embedded human tissue specimens. ...Hybridization (FISH) Enumeration Systems.â See § 866.1(e)...

2009-04-01

231

Phylogenetic Composition, Spatial Structure, and Dynamics of Lotic Bacterial Biofilms Investigated by Fluorescent in Situ Hybridization and Confocal Laser Scanning Microscopy  

Microsoft Academic Search

The phylogenetic composition, three-dimensional structure and dynamics of bacterial communities in river biofilms generated\\u000a in a rotating annular reactor system were studied by fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). Biofilms grew on independently removable polycarbonate\\u000a slides exposed in the reactor system with natural river water as inoculum and sole nutrient and carbon source. The microbial

W. Manz; K. Wendt-Potthoff; T. R. Neu; U. Szewzyk; J. R. Lawrence

1999-01-01

232

6q deletion detected by fluorescence in situ hybridization using bacterial artificial chromosome in chronic lymphocytic leukemia.  

PubMed

Deletions of the long arm of chromosome 6 are known to occur at relatively low frequency (3-6%) in chronic lymphocytic leukemia (CLL), and they are more frequently observed in 6q21. Few data have been reported regarding other bands on 6q involved by cytogenetic alterations in CLL. The cytogenetic study was performed in nuclei and metaphases obtained after stimulation with a combination of CpG-oligonucleotide DSP30 and interleukin-2. Four bacterial artificial chromosome (BAC) clones mapping regions in bands 6q16, 6q23, 6q25, 6q27 were used as probes for fluorescence in situ hybridization in 107 CLL cases in order to analyze the occurrence and localization of 6q aberrations. We identified 11 cases (10.2%) with 6q deletion of 107 patients studied with CLL. The trends of survival curves and the treatment-free intervals (TFI) of patients with deletion suggest a better outcome than the other cytogenetic risk groups. We observed two subgroups with 6q deletion as the sole anomaly: two cases with 6q16 deletion, and three cases with 6q25.2-27 deletion. There were differences of age, stage, and TFI between both subgroups. By using BAC probes, we observed that 6q deletion has a higher frequency in CLL and is linked with a good prognosis. In addition, it was observed that the deletion in 6q16 appears to be the most frequent and, if present as the only abnormality, it could be associated with a most widespread disease. PMID:23560441

Dalsass, Alessia; Mestichelli, Francesca; Ruggieri, Miriana; Gaspari, Paola; Pezzoni, Valerio; Vagnoni, Davide; Angelini, Mario; Angelini, Stefano; Bigazzi, Catia; Falcioni, Sadia; Troiani, Emanuela; Alesiani, Francesco; Catarini, Massimo; Attolico, Immacolata; Scortechini, Ilaria; Discepoli, Giancarlo; Galieni, Piero

2013-07-01

233

Fluorescence in situ hybridization as an ancillary tool in the diagnosis of ambiguous melanocytic neoplasms: a review of 804 cases.  

PubMed

Previous studies have demonstrated the utility of fluorescence in situ hybridization (FISH) as an ancillary method in the diagnostic workup of histopathologically ambiguous melanocytic neoplasms. A combination of probes targeting 3 loci on chromosome 6 and 1 on 11q has been reported to distinguish unequivocal melanomas and nevi with a sensitivity and specificity of 87% and 96%, respectively. However, information on how FISH should be integrated into routine clinical testing is limited. We report our experience of FISH testing of 804 ambiguous melanocytic lesions performed as part of routine workup at University of California, San Francisco. The main category (47% of all cases) for which FISH testing was requested was Spitz tumors. Other categories included the distinction of possible melanoma from combined nevi (9%), acral or mucosal nevi (9%), Clark/dysplastic nevi (7%), and blue or deep penetrating nevi (6%) and to assess the possibility of nevoid melanoma (4%). Of the ambiguous tumors successfully tested, 88% received a more definitive benign or malignant final diagnosis. Of the 630 cases that tested negative by FISH, the final diagnosis was benign in 489 (78%) cases, ambiguous in 91 cases (14%), and malignant in 50 cases (8%). A positive FISH result was observed in 124 cases, with a final diagnosis of melanoma in 117 (94%). One (1%) FISH-positive case had an equivocal final diagnosis, and 6 (5%) were interpreted, despite the positive FISH result, as melanocytic nevi. We conclude that FISH testing can help reduce the number of equivocal diagnoses in ambiguous melanocytic neoplasms, in particular if FISH testing is positive, and discuss the challenges and limitations of FISH in clinical practice. PMID:24618603

North, Jeffrey P; Garrido, Maria C; Kolaitis, Nicholas A; LeBoit, Philip E; McCalmont, Timothy H; Bastian, Boris C

2014-06-01

234

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

PubMed Central

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.

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

2013-01-01

235

Use of a modified fluorescent in situ hybridization procedure to improve the identification of Streptococcus pneumoniae in blood cultures.  

PubMed

Streptococcus pneumoniae is an important causative agent for bacteremia. Fluorescent in situ hybridization (FISH) is a helpful molecular technique for the rapid identification of S. pneumoniae in positive blood cultures. There are many reports concerning the application of an enzymatic treatment with lysozyme in the FISH procedure for partial cell wall digestion of S. pneumoniae. However, this study was aimed to test the FISH procedure without enzymatic treatment for the identification of S. pneumoniae in blood culture specimens. Seventy-seven positive blood culture specimens containing Gram-positive cocci were examined by both the conventional laboratory methods and FISH. Detection of S. pneumoniae was performed by two FISH procedures: one procedure was performed with an enzymatic treatment step and the other one was done without enzymatic treatment. In addition, the specimens were tested by the FISH procedure with enzymatic treatment to detect Streptococcus pyogenes and Enterococcus. The specificity of FISH in comparison with conventional culture methods was 100%. The sensitivity of the FISH procedure with enzymatic treatment for the detection of S. pneumoniae was 90%, whereas, the sensitivity of the FISH procedure without enzymatic treatment was 100%. In fact, by omission of enzymatic treatment, detection of S. pneumoniae was improved in 6 specimens. The results of the FISH and culture methods for the detection of S. pyogenes and Enterococcus were compatible. Altogether, FISH procedure without enzymatic treatment step seems to improve the detection of S. pneumoniae in some cases. Thus, for successful detection of S. pneumoniae, we suggest the application of both FISH procedures (the procedure with enzymatic treatment and the procedure without enzymatic treatment) for each blood culture specimen. PMID:24060554

Tajbakhsh, Saeed; Gharibi, Somayyeh; Zandi, Keivan; Yaghobi, Ramin

2013-09-01

236

Sequential multiple probe fluorescence in-situ hybridization analysis of human oocytes and polar bodies by combining centromeric labelling and whole chromosome painting  

Microsoft Academic Search

The incidence of chromosomal aneuploidy was analysed in 104 unfertilized human oocytes and 56 first polar bodies using a double-label fluorescence in-situ hybridization (FISH) procedure. Combinations of centromeric (or locus-specific) DNA probes and whole chromosome painting probes for chromosomes 9, 13, 16, 18, 21 and X were applied on oocyte preparations, in a sequential FISH protocol. This combined approach allowed

T. Anahory; B. Andreo; G. Regnier-Vigouroux; J. P. Soulie; M. Baudouin; J. Demaille; F. Pellestor

2003-01-01

237

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

Microsoft Academic Search

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

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

1997-01-01

238

Assessment of chromosomal abnormalities in sperm of infertile men using sperm karyotyping and multicolour fluorescence in situ hybridization (FISH)  

SciTech Connect

Individuals with male factor infertility resulting from idiopathic oligo-, astheno- or teratozoospermia are frequently offered IVF in an attempt to increase their chances of having a child. A concern remains whether these infertile males have an elevated risk of transmitting chromosomal abnormalities to their offspring. Sperm chromosomal complements from these men were assayed using the human sperm/hamster oocyte fusion system and fluorescence in situ hybridization (FISH) on sperm nuclei. For each of 5 infertile patients, 100 sperm karyotypes were analyzed and multicolour FISH analysis was performed on a minimum of 10,000 sperm nuclei for each chromosome-specific DNA probe for chromosomes 1 (pUC1.77), 12 (D12Z3), X (XC) and Y (DYZ3). As a group, the infertile patients showed increased frequencies of both numerical ({chi}{sup 2}=17.26, {proportional_to} <0.001) and total abnormalities ({chi}{sup 2}=7.78, {proportional_to} <0.01) relative to control donors when assessed by sperm karyotypes. Analysis of sperm nuclei by FISH indicated a significant increase in the frequency of disomy for chromosome 1 in three of the five patients as compared to control donors ({chi}{sup 2}>8.35, {proportional_to} <0.005). In addition, the frequency of XY disomy was significantly higher in four of the five patients studied by FISH ({chi}{sup 2}>10.58, {proportional_to}<0.005), suggesting that mis-segregation caused by the failure of the XY bivalent to pair may play a role in idiopathic male infertility.

Moosani, N.; Martin, R.H. [Alberta Children`s Hospital and Univ. of Calgary (Canada)

1994-09-01

239

Multiplex fluorescence in situ hybridization identifies novel rearrangements of chromosomes 6, 15, and 18 in primary uveal melanoma.  

PubMed

Uveal melanomas are the commonest ocular tumour of adults and are characterized by reproducible alterations of chromosomes 1, 3, 6 and 8. These alterations are of prognostic relevance and have also be shown to correlate to high risk and low risk metastatic categories of uveal melanoma as defined by micro-array analysis. It is, however, possible that a catalogue of relevant genetic alterations, involving gene rearrangement rather than amplification, have as yet eluded identification. To address this point we examined 14 primary uveal melanomas, using 24 colour multiplex fluorescence in situ hybridization (M-FISH). All tumours were karyotyped following G-Banding, and M-FISH was performed to confirm and clarify the identity of abnormal chromosomes. M-FISH data were obtained from all tumours and was able to establish the nature of most abnormalities not fully characterized by cytogenetics. Abnormalities of chromosome 6 were far more frequent than previously indicated, in approximately 70% of cases, indicating they have been substantially underrepresented in past studies of uveal melanoma. Spindle melanomas were found to have novel rearrangements affecting in particular chromosomes 6, 15 and 18, suggesting that juxtaposition of genes through translocational events may play a role in the development of some uveal melanomas. In conclusion, this study is the largest of primary uveal melanoma analysed by M-FISH and indicates that alterations of chromosome 6 have previously been underestimated. Furthermore spindle melanomas are prone to rearrangements affecting chromosomes 6, 15 and 18, which may relate to early changes in uveal melanoma development or associate with those melanomas of a more differentiated status. PMID:16684523

Sisley, Karen; Tattersall, Nicola; Dyson, Michael; Smith, Kath; Mudhar, Hardeep S; Rennie, Ian G

2006-09-01

240

A simple method for simultaneous R- or G-banding and fluorescence in situ hybridization of small single-copy genes  

Microsoft Academic Search

A significant improvement in fluorescence in situ hybridization, enabling the detection of single-copy genes as small as 500 bp directly on banded chromosomes, is presented. The induction of chromosome banding, which does not require additional handling or any system of amplification, is obtained simply by using an alkaline (pH 11) p-phenylenediamine anti-fade solution. As the banding produced is related to

N. Lemieux; B. Dutrillaux; E. Viegas-Péquignot

1992-01-01

241

A high-resolution cytogenetic map of human chromosome 12: localization of 195 new cosmid markers by direct R-banding fluorescence in situ hybridization.  

PubMed

We have constructed a high-resolution cytogenetic map of human chromosome 12 with 195 newly isolated cosmids by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 195 clones were evenly distributed throughout chromosome 12, but sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average map distance of 0.73 Mb on bands can, in conjunction with a genetic linkage map, facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Moreover, the cytogenetic mapping data provide starting points for establishing contig maps with cosmid clones and yeast artificial chromosomes. PMID:8225322

Takahashi, E; Koyama, K; Hitomi, A; Nakamura, Y

1993-10-01

242

A high-resolution cytogenetic map of human chromosome 5: localization of 206 new cosmid markers by direct R-banding fluorescence in situ hybridization.  

PubMed

We have constructed a high-resolution cytogenetic map of human chromosome 5 with 206 new cosmid clones by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 206 clones were evenly distributed throughout chromosome 5, although they were sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average distance of nearly 1 Mb will serve as important resource for construction of a genetic linkage map, which is essential for positional cloning of responsible genes. Moreover, these mapping data provide many useful landmarks for the construction of contig maps of chromosome 5, as required in the Human Genome Project. PMID:8406458

Takahashi, E; Hitomi, A; Nakamura, Y

1993-07-01

243

Analysis of sex-mismatched human corneal transplants by fluorescence in situ hybridization of the sex-chromosomes.  

PubMed

The fate of the cells of corneal transplants has been controversial from the early days of keratoplasty. Various methods such as histological evaluation, radiolabeling of donor cells or Barr-body analysis have been applied to clarify the issue. However, the question whether the transplanted cells are replaced or survive, remains unsolved. In this study, we applied fluorescence in situ hybridization (FISH) of the X- and Y-chromosomes in paraffin sections of explanted sex-mismatched corneal transplants to distinguish between host and donor cells. Fourteen sex-mismatched cases with various reasons for explantation and different postoperative time intervals ranging from 11 months to 30 years were analysed. We found that all cell types, including epithelium, keratocytes and endothelial donor cells were replaced in most cases as early as 1 year after transplantation. In three cases, however, up to 26% of donor keratocytes were still detected up to 4.5 years after transplantation, demonstrating a certain individual variability in the process of replacement. Further studies must show if the extent and timing of donor cell replacement in clinically successful, totally clear transplants is different. Our results are in keeping with the phenomenon of recurrences of corneal dystrophies in the graft, the significant postoperative decline of the endothelial cell density, the fact that typical graft rejections usually take place within 1-2 years postoperatively and that relatively late rejections can occur in rare cases probably due to some surviving stromal keratocytes. Donor cell replacement is a special feature of corneal transplants when compared with other kinds of organ transplants and might be due to the presence of the same tissue type in the immediate neighbourhood of the graft. PMID:10079142

Wollensak, G; Green, W R

1999-03-01

244

Fluorescence in situ Hybridization method using Peptide Nucleic Acid probes for rapid detection of Lactobacillus and Gardnerella spp.  

PubMed Central

Background Bacterial vaginosis (BV) is a common vaginal infection occurring in women of reproductive age. It is widely accepted that the microbial switch from normal microflora to BV is characterized by a decrease in vaginal colonization by Lactobacillus species together with an increase of Gardnerella vaginalis and other anaerobes. Our goal was to develop and optimize a novel Peptide Nucleic Acid (PNA) Fluorescence in situ Hybridization assay (PNA FISH) for the detection of Lactobacillus spp. and G. vaginalis in mixed samples. Results Therefore, we evaluated and validated two specific PNA probes by using 36 representative Lactobacillus strains, 22 representative G. vaginalis strains and 27 other taxonomically related or pathogenic bacterial strains commonly found in vaginal samples. The probes were also tested at different concentrations of G. vaginalis and Lactobacillus species in vitro, in the presence of a HeLa cell line. Specificity and sensitivity of the PNA probes were found to be 98.0% (95% confidence interval (CI), from 87.8 to 99.9%) and 100% (95% CI, from 88.0 to 100.0%), for Lactobacillus spp.; and 100% (95% CI, from 92.8 to 100%) and 100% (95% CI, from 81.5 to 100.0%) for G. vaginalis. Moreover, the probes were evaluated in mixed samples mimicking women with BV or normal vaginal microflora, demonstrating efficiency and applicability of our PNA FISH. Conclusions This quick method accurately detects Lactobacillus spp. and G. vaginalis species in mixed samples, thus enabling efficient evaluation of the two bacterial groups, most frequently encountered in the vagina.

2013-01-01

245

Clear cell papillary renal cell carcinoma with angiomyomatous stroma: a histological, immunohistochemical, and fluorescence in situ hybridization study.  

PubMed

Clear cell papillary renal cell carcinoma (CCPRCC) is a novel tumor entity that was recently recognized as a new distinct epithelial tumor within the current classification system. Nonclassic morphologic variants have rarely been reported. We present six challenging cases of CCPRCC with prominent (>75 %) tubular, acinar, and/or solid component and angioleiomyomatous stroma. The tumors lacked well-organized papillary architecture. All tumors had a variously thick capsule formed by a layer of bands of smooth muscle. The leiomyomatous tissue often entirely encased patches of tubular structures, or it formed only small leiomyomatous islands within the epithelial component. There was a remarkable relationship between the vascular network and the epithelial component in the sense that every single tubule or acinus was associated with a fine capillary network, with the capillaries intimately surrounding the tubular or acinar circumference. CCPRCC with variant morphology expressed carbonic anhydrase IX (CA-IX) in cup-shaped distribution. In addition, the tumor cells stained positive for cytokeratin 34betaE12, CK7, and vimentin. Renal cell carcinoma (RCC), P504s/AMACR, Melan A, and HMB45 were negative in tumor cells in all cases examined. Fluorescence in situ hybridization studies showed the presence of a normal copy number for chromosomes 7, 17, 3q, and 3p. CCPRCC with variant morphology seems to have a favorable prognosis. In the current series, tumor stage was low at presentation, and none of the patients had local recurrence or metastatic disease. The distinction between CCPRCC with variant morphology and clear cell RCC is critical because no case of CCPRCC has behaved aggressively. PMID:24771120

Alexiev, Borislav A; Thomas, Carrie; Zou, Ying S

2014-06-01

246

Fluorescence In Situ Hybridization: A Sensitive Method for Trisomy 8 Detection in Bone Marrow Specimens  

Microsoft Academic Search

RISOMY 8 is a common cytogenetic abnormality T found in the bone marrow (BM) of patients with myeloproliferative disorders (MPD), myelodysplastic syn- dromes (MDS), or acute nonlymphocytic leukemia (ANLL).1-5 Using conventional cytogenetic methods in which 20 to 30 metaphase cells are typically examined, it is possible to find 2 or more metaphases with trisomy 8 in many of these patients.

Robert B. Jenkins; Michelle M. Le Beau; William J. Kraker; Thomas J. Borell; Paul G. Stalboerger; Elizabeth M. Davis; Lolita Penland; Anthony Fernald; Rafael Espinosa; Daniel J. Schaid; Gordon W. Dewald

1992-01-01

247

Rapid differentiation of Francisella species and subspecies by fluorescent in situ hybridization targeting the 23S rRNA  

PubMed Central

Background Francisella (F.) tularensis is the causative agent of tularemia. Due to its low infectious dose, ease of dissemination and high case fatality rate, F. tularensis was the subject in diverse biological weapons programs and is among the top six agents with high potential if misused in bioterrorism. Microbiological diagnosis is cumbersome and time-consuming. Methods for the direct detection of the pathogen (immunofluorescence, PCR) have been developed but are restricted to reference laboratories. Results The complete 23S rRNA genes of representative strains of F. philomiragia and all subspecies of F. tularensis were sequenced. Single nucleotide polymorphisms on species and subspecies level were confirmed by partial amplification and sequencing of 24 additional strains. Fluorescent In Situ Hybridization (FISH) assays were established using species- and subspecies-specific probes. Different FISH protocols allowed the positive identification of all 4 F. philomiragia strains, and more than 40 F. tularensis strains tested. By combination of different probes, it was possible to differentiate the F. tularensis subspecies holarctica, tularensis, mediasiatica and novicida. No cross reactivity with strains of 71 clinically relevant bacterial species was observed. FISH was also successfully applied to detect different F. tularensis strains in infected cells or tissue samples. In blood culture systems spiked with F. tularensis, bacterial cells of different subspecies could be separated within single samples. Conclusion We could show that FISH targeting the 23S rRNA gene is a rapid and versatile method for the identification and differentiation of F. tularensis isolates from both laboratory cultures and clinical samples.

2010-01-01

248

Assignment of the human homologue of the drosophila cut homeobox gene (CUTL1) to band 7q22 by fluorescence in situ hybridization  

SciTech Connect

Three cDNA probes that were used for the mapping. The regions covered by these probes comprise nucleotides 2492 to 5374 for probe 1, 2492 to 3936 for probe 2, and 3936 to 5374 for probe 3. The chromosomal localization of the CUTL1 gene was determined by FISH (fluorescence in situ hybridization). Probes were labeled by nick-translation using biotin-11-dUTP. After hybridization, slides were rinsed once with 2 x SSC. Probes were visualized by an indirect immunofluorescence system without any amplification.

Lemieux, N.; Zhang, X.X. [Universite de Montreal, Quebec (Canada)] [Universite de Montreal, Quebec (Canada); Dufort, D. [McGill Univ, Montreal (Canada)] [and others] [McGill Univ, Montreal (Canada); and others

1994-11-01

249

Clinical utility of fluorescence in situ hybridization (FISH) in nonbrainstem glioblastomas of childhood  

Microsoft Academic Search

Astrocytic gliomas are the most common pediatric brain tumors; however, nonbrainstem glioblastomas are extremely rare compared with their adult counterparts. Little information is available on the clinical significance of various molecular markers in pediatric grade IV astrocytomas. The current study was focused on the molecular analysis and clinico-pathological correlations in a set of 44 tumor samples obtained from pediatric patients

Andrey Korshunov; Regina Sycheva; Sergey Gorelyshev; Andrey Golanov

2005-01-01

250

Correlations between arsenic in Maine groundwater and microbial populations as determined by fluorescence in situ hybridization  

Microsoft Academic Search

Arsenic is known to cause serious health effects when consumed in drinking water. In the state of Maine, approximately half of the population relies on private groundwater wells for their drinking water. Of those wells, as many as 13% may contain arsenic levels above the current EPA maximum contaminant level of 10?gl?1. Microorganisms can potentially contribute to arsenic release into

Jennifer M. Weldon; Jean D. MacRae

2006-01-01

251

Fluorescence In Situ Hybridization and Immunohistochemistry as Diagnostic Methods for ALK Positive Non-Small Cell Lung Cancer Patients  

PubMed Central

Background Anaplastic Lymphoma Kinase (ALK) positivity represents a novel molecular target in a subset of Non-Small Cell Lung Cancers (NSCLC). We explore Fluorescence in situ Hybridization (FISH) and Immunohistochemistry (IHC) as diagnostic methods for ALK positive patients and to describe its prevalence and outcomes in a population of NSCLC patients. Methods NSCLC patients previously screened for Epidermal Growth Factor Receptor (EGFR) at our institution were selected. ALK positive patients were identified by FISH and the value of IHC (D5F3) was explored. Results ninety-nine patients were identified. Median age was 61.5 years (range 35–83), all were caucasians, eighty percent were adenocarcinomas, fifty-one percent were male and thirty-eight percent were current smokers. Seven (7.1%) patients were ALK positive by FISH, thirteen (13.1%) were EGFR mutant, and 65 (65.6%) were negative/Wild Type (WT) for both ALK and EGFR. ALK positivity and EGFR mutations were mutually exclusive. ALK positive patients tend to be younger than EGFR mutated or wt patients. ALK positive patients were predominantly never smokers (71.4%) and adenocarcinoma (71.4%). ALK positive and EGFR mutant patients have a better outcome than negative/WT. All patients with ALK FISH negative tumours were negative for ALK IHC. Out of 6 patients positive for ALK FISH with more tissue available, 5 were positive for ALK IHC and 1 negative. Conclusions ALK positive patients represent 7.1% of a population of selected NSCLC. ALK positive patients have different clinical features and a better outcome than EGFR WT and ALK negative patients. IHC is a promising method for detecting ALK positive NSCLC patients.

Martinez, Pablo; Hernandez-Losa, Javier; Cedres, Susana; Castellvi, Josep; Martinez-Marti, Alex; Tallada, Natalia; Murtra-Garrell, Nuria; Navarro-Mendivill, Alejandro; Rodriguez-Freixinos, Victor; Canela, Mercedes; Ramon y Cajal, Santiago; Felip, Enriqueta

2013-01-01

252

Localization of a candidate colon tumor-suppressor gene (DRA) to 7q22-q31. 1 by fluorescence in situ hybridization  

SciTech Connect

The authors have previously reported that the DRA gene is located on chromosome 7. This assignment was based on Southern blot hybridization of a DRA cDNA to genomic DNA from rodent-human somatic cell hybrids. In this report, they localize the DRA gene to chromosome band 7q22-q31.1 by fluorescence in situ hybridization (FISH) with a full-length (2.9 kb) cDNA as probe. Metaphase spreads from normal human lymphocytes were prepared according to the method of Fan et al. The cDNA clone 611C was labeled with biotin-11-dUTP using a nick-translation kit (Oncor) followed by purification on a Sephadex G50-fine column. FISH and detection of immunofluorescence were performed according to the technique of Pinkel et al. with minor modifications. The chromosome preparations were stained with both diamidino-2-phenylindole and propidium iodide (Oncor) and observed with a Zeiss Axiophot fluorescence microscope. Hybridization was detected on chromosome 7 in 22 of 47 spreads examined. Of 89 fluorescent signals on all chromosomes, 44 (49%) were located on 7q. All signals on chromosome 7 appeared to be located at 7q22-q31.1. Hybridization is in the vicinity of the met protooncogene locus at 7q31.

Taguchi, T.; Testa, J.R. (Fox Chase Cancer Center, Philadelphia, PA (United States)); Papas, T.S.; Schweinfest, C. (Medical Univ. of South Carolina, Charleston, SC (United States))

1994-03-01

253

Combination of Fluorescence In Situ Hybridization with Staining Techniques for Cell Viability and Accumulation of PHA and polyP in Microorganisms in Complex Microbial Systems  

NASA Astrophysics Data System (ADS)

Fluorescence in situ hybridization (FISH) can be combined with a number of staining techniques to reveal the relationships between the microorganisms and their function in complex microbial systems with a single-cell resolution. In this chapter, we have focused on staining methods for intracellular storage compounds (polyhydroxyalkanoates, polyphosphate) and a measure for cell viability, reduction of the tetrazolium-based redox stain CTC. These protocols are optimized for the study of microorganisms in waste-water treatment (activated sludge and biofilms), but they may also be used with minor modifications in many other ecosystems.

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

254

Quantitative real-time PCR and fluorescence in situ hybridization approaches for enumerating Brevundimonas diminuta in drinking water.  

PubMed

Brevundimonas diminuta is a small Gram-negative bacterium used for validation of membranes and filters used in the pharmaceutical and drinking water treatment industries. Current assays are time consuming, nonselective, and may be subject to interference by competing indigenous microorganisms. The focus of this study is to develop rapid and specific enumeration methodologies for B. diminuta. Quantitative real-time polymerase chain reaction (qPCR) and fluorescence in situ hybridization (FISH) assays were developed based on the gyrB (1,166 bp) and rpoD (829 bp) gene sequences of B. diminuta ATCC 19146. Species-specific primers and probes were designed, and a 100-200 bp segment of each gene was targeted in the qPCR studies. For both the qPCR and FISH assays, an internal 25 bp sequence was selected for use as a TaqMan probe (labeled with 6-FAM and a Black Hole Quencher). Probe specificity studies, conducted against Gram-negative and Gram-positive reference strains as well as environmental strains, revealed high specificity of the primer/probe pairs to B. diminuta. Sensitivities of the qPCR reactions using purified genomic DNA from B. diminuta were determined to be 0.89 pg for rpoD and 8.9 pg for gyrB. The feasibility of using whole-cell B. diminuta suspensions directly with the rpoD qPCR protocol was also evaluated. The greatest sensitivity observed for B. diminuta was 1 x 10(3) colony forming units (CFU) per mL when tryptic soy broth was used as the growth medium. When compared with direct microscopic enumeration using a 5' 6-FAM FISH probe, traditional plating methods showed significant underestimation of B. diminuta concentration (P = 0.01) when this organism was cultivated in saline lactose broth. The results of this investigation demonstrate that qPCR and FISH are effective methods for rapid (<4 h) enumeration of B. diminuta and may be viable alternatives to plating when validating drinking water filtration systems. PMID:20495940

Donofrio, Robert S; Bestervelt, Lorelle L; Saha, Ratul; Bagley, Susan T

2010-09-01

255

The detection of contiguous gene deletions at the neurofibromatosis 1 locus with fluorescence in situ hybridization  

Microsoft Academic Search

Neurofibromatosis type 1 (NFl) is a common genetic disorder characterized primarily by the development of multiple neurofibromas and pigmentary changes. The recent identification of contiguous gene deletions in NF1 a previously unrecognized molecular basis for this disorder, raises important questions regarding deletion frequency in the patient population and the role that contiguous genes may play in the physical manifestations of

K. A. Leppig; D. Viskochil; S. Neil; A. Rubenstein; V. P. Johnson; X. L. Zhu; A. R. Brothman; K. Stephens

1996-01-01

256

Identification of two Skeletonema costatum -like diatoms by fluorescence in situ hybridization  

Microsoft Academic Search

A harmful algae bloom (HAB) is a dense aggregation of algae in a marine or aquatic environment that can result in significant\\u000a environmental problems. To forecast the occurrence of HAB, development of a rapid and precise detection method is urgently\\u000a required. In this study, two Skeletonema costatum-like diatoms (SK-1 and SK-2), were identified morphologically under a light microscope, and detected

Baoyu Zhang; Guofu Chen; Guangce Wang; Douding Lu

2010-01-01

257

Combined Fluorescent-Chromogenic In Situ Hybridization for Identification and Laser Microdissection of Interphase Chromosomes  

PubMed Central

Chromosome territories constitute the most conspicuous feature of nuclear architecture, and they exhibit non-random distribution patterns in the interphase nucleus. We observed that in cell nuclei from humans with Down Syndrome two chromosomes 21 frequently localize proximal to one another and distant from the third chromosome. To systematically investigate whether the proximally positioned chromosomes were always the same in all cells, we developed an approach consisting of sequential FISH and CISH combined with laser-microdissection of chromosomes from the interphase nucleus and followed by subsequent chromosome identification by microsatellite allele genotyping. This approach identified proximally positioned chromosomes from cultured cells, and the analysis showed that the identity of the chromosomes proximally positioned varies. However, the data suggest that there may be a tendency of the same chromosomes to be positioned close to each other in the interphase nucleus of trisomic cells. The protocol described here represents a powerful new method for genome analysis.

Paz, Nerea; Zabala, Amaia; Royo, Felix; Garcia-Orad, Africa; Zugaza, Jose L.; Parada, Luis A.

2013-01-01

258

Rapid method for measuring clastogenic fingerprints using fluorescence in situ hybridization  

DOEpatents

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.

Lucas, Joe N. (San Ramon, CA) [San Ramon, CA

2000-01-01

259

Analysis of amniotic fluid specimens for common chromosome disorders using interphase fluorescence in situ hybridization  

Microsoft Academic Search

Objective: The aim of the study was to examine the usage of multi colour FISH technology as an adjunct to con- ventional cytogenetics for the prenatal diagnosis of aneuploidy in interphase nuclei from high risk pregnancies. Methods: Amniotic fluid samples were collected for interphase FISH analysis using DNA probes for chromo- somes 13, 18, 21, X and Y. All the

Tariq Moatter; Zahida Khilji; Farzana Murad; Shama Munim

260

Diagnosis of a constitutional five-chromosome rearrangement by fluorescent in situ hybridization (FISH)  

SciTech Connect

Complex chromosomal rearrangements are structural rearrangements involving at least three chromosomes and three or more chromosome breakpoints. Such karyotypes are often acquired during cancer multi-step development and in chromosome instability syndromes. However, extremely rare constitutional forms have been reported, most of which are incompatible with life. We present a 2-year-old female with de novo complex rearrangement consisting of five chromosomes and nine breakpoints. Clinical evaluation at two years of age revealed a weight of 5 kg, length of 66 cm, and had circumference of 38 cm, all below the 5th percentile, microcephaly, trigonocephaly, epicanthal folds, inguinal hernia, left clubfoot, hypertonicity, and developmental delay. The neurological examination revealed chorea-acanthocytosis and psychomotor delay. Cultured lymphocytes and fibroblasts revealed a karyotype consisting of five derivative chromosomes. The metaphases were further analyzed by FISH using chromosome-specific libraries and telomeric probes in order to delineate the composition of the rearranged chromosomes; FISH results demonstrated a karyotype of: 46,XX,1pter{r_arrow}1q25::1q42.1{r_arrow}1qter, 2pter{r_arrow}q32.3::1q32.3{r_arrow}2q41::2q37.3{r_arrow}2qter, 7qter{r_arrow}7q21.2::6q22.3{r_arrow}6qter::1q31{r_arrow}1q32.3::6p23{r_arrow}6q22.3, 7pter{r_arrow}7q21.1::6p23{r_arrow}6pter, 2q33{r_arrow}2q37, 1::9p21{r_arrow}9qter. This analysis demonstrates the usefulness of FISH in characterizing complex chromosome rearrangements otherwise difficult to correctly interpret using classical cytogenetics alone.

Tsien, F.; Shapira, E. [Tulane Univ. School of Medicine, New Orleans, LA (United States); Carvalho, T. [Hospital Sarah Kubitschek, Brasilia (Brazil)] [and others

1994-09-01

261

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

SciTech Connect

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.

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

1994-09-01

262

Gene-specific fluorescence in-situ hybridization analysis on tissue microarray to refine the region of chromosome 20q amplification in melanoma.  

PubMed

Several comparative genomic hybridization studies provide evidence for overrepresentation of the long arm of chromosome 20 in malignant melanoma. These studies also suggest that chromosome 20q contains genes that may contribute to melanoma pathogenesis. To refine the region of 20q amplification and to identify potential candidate genes involved in melanoma or even in melanoma progression from these regions, we combined fluorescence in-situ hybridization with MYBL2, ZNF217, CYP24 and STK6 specific probes (chromosomal region 20q13.1-q13.2) with high-throughput tissue microarray consisting of 280 primary melanomas and melanoma metastases. Low-level amplification ranging from 0.5 to 2.0% was detected for the tumor-related genes of interest. Higher frequencies of gain when compared with amplification were detected for MYBL2, ZNF217, CYP24 and STK6. Aneusomy of centromere 20 was observed in 29.9% of the analyzed tumors. A significantly higher frequency of ZNF217, CYP24 and STK6 total copy-number increase, as well as aneusomy of centromere 20, was found in the group of metastases when compared with the group of primary melanomas. Despite the technological advantage of fluorescence in-situ hybridization on tissue microarray, which allows refining regions of amplification, we were not able to recognize any of the MYBL2, ZNF217, CYP24 and STK6 genes as a particular relevant gene for melanoma tumorigenesis. PMID:17235240

Koynova, Denitsa K; Jordanova, Ekaterina S; Milev, Angel D; Dijkman, Remco; Kirov, Krassimir S; Toncheva, Draga I; Gruis, Nelleke A

2007-02-01

263

Molecular and fluorescence in situ hybridization analysis of a 10;11 rearrangement in a case of infant acute monocytic leukemia.  

PubMed

Fluorescence in situ hybridization (FISH) analysis in a case of infant acute monocytic leukemia M5 revealed a complex rearrangement between chromosomes 10 and 11, leading to the disruption of the MLL gene. Using two painting probes for chromosomes 10 and 11 and a specific probe for the MLL gene localized on 11q23, we observed a paracentric inversion of the 11q13-q23 fragment translocated to 10p12. Molecular analysis showed that AF10 localized on 10p12 was the fusion partner gene of MLL in this rearrangement (10;11). This report underlined the usefulness of FISH and molecular techniques in identifying complex rearrangements. PMID:12127405

Roll, Patrice; Zattara-Cannoni, Hélčne; Bustos-Bernard, Marie Christine; Curtillet, Catherine; Michel, Gérard; Vagner-Capodano, Anne-Marie

2002-06-01

264

Identification of a ring chromosome as a ring 8 using fluorescent in situ hybridization (FISH) in a child with multiple congenital anomalies  

SciTech Connect

We read with interest the report by Melnyk and Dewald of a small supernumerary ring chromosome 8 identified by fluorescence in situ hybridization (FISH) in a child with developmental delay and minor anomalies. Although ring chromosomes resulting in loss of parts of chromosome 8 have been reported, Melnyk and Dewald reported the first small ring chromosome 8 diagnosed by FISH. Previously nonsatellited markers derived from chromosomes 1, 3, 6, 9, 11, 13-16, 18, 20, 21, and X have been identified using FISH. Their study illustrated the value of FISH techniques in identifying the chromosomal source of markers or rings.

Butler, M.G.; Roback, E.W.; Allen, G.A. [Genetic Associates, Nashville, TN (United States)

1995-07-03

265

Fluorescence in situ hybridization analysis of two blastomeres from day 3 frozen-thawed embryos followed by analysis of the remaining embryo on day 5  

Microsoft Academic Search

BACKGROUND: Chromosomal mosaicism in human embryos may give rise to false\\u000a positive or false negative results in preimplantation genetic diagnosis\\u000a for aneuploidy screening (PGD-AS). Therefore, we have investigated whether\\u000a the results obtained from a 2-cell biopsy of frozen-thawed embryos and\\u000a fluorescence in situ hybridization (FISH) analysis are representative for\\u000a the chromosome constitution of the remaining embryo on day 5. METHODS:

E. B. Baart; Opstal van A. R. M; F. J. Los; B. C. J. M. Fauser; E. Martini

2004-01-01

266

A high-resolution cytogenetic map of human chromosome 2: localization of 434 cosmid markers by direct R-banding fluorescence in situ hybridization.  

PubMed

We have constructed a high-resolution cytogenetic map of human chromosome 2 with 434 newly isolated cosmid markers by means of direct R-banding fluorescence in situ hybridization. Two markers were mapped to the centromeric region, 173 to the short arm, and 259 to the long arm. The clones were evenly distributed along the entire chromosome, although a tendency toward clustering in R-positive bands was observed. The mapped cosmids provide useful landmarks for construction of a contig map and for positional cloning of cancer-associated genes, as well as genes responsible for hereditary diseases. PMID:7956348

Takahashi, E; Koyama, K; Hirai, M; Itoh, H; Nakamura, Y

1995-01-01

267

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

PubMed Central

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 52 mycobacterial culture isolates. Results were compared to conventional genotypic identification with amplification-based methods. All isolates (M. tuberculosis complex, n = 24; M. avium, n = 7; M. kansasii, n = 1) were correctly identified by FISH. In addition, the technique was used successfully for visualization of mycobacteria in biopsies from infected humans or animals. In conclusion, PNA-FISH is a fast and accurate tool for species-specific identification of culture-grown mycobacteria and for direct visualization of these organisms in tissue sections. It may be used successfully for both research and clinical microbiology.

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

2006-01-01

268

A high-resolution cytogenetic map of human chromosome 9: Localization of 203 new cosmid markers by direct R-banding fluorescence in situ hybridization  

SciTech Connect

A high-resolution cytogenetic map of human chromosome 9 was constructed with 203 newly isolated cosmid markers by direct R-banding fluorescence in situ hybridization. The clones were localized preferentially to R-positive bands throughout chromosome 9. Although the number of clones was roughly proportional to the R-band size, many more clones (ca. 80) were mapped at the q34 region compared to its physical size. Based on these cytogenetic mapping data, one can establish physical contig maps with cosmids and yeast artificial chromosomes and a genetic linkage map essential for positional cloning of responsible genes. 13 refs., 2 figs., 1 tab.

Takahashi, Ei-ichi; Hitomi, Akitsu; Itoh, Hiroko (National Institute of Radiological Sciences, Chiba (Japan)); Koyama, Kumiko; Nakamura, Yusuke (Cancer Institute, Tokyo (Japan))

1994-01-15

269

A high-resolution cytogenetic map of human chromosome 9: localization of 203 new cosmid markers by direct R-banding fluorescence in situ hybridization.  

PubMed

A high-resolution cytogenetic map of human chromosome 9 was constructed with 203 newly isolated cosmid markers by direct R-banding fluorescence in situ hybridization. The clones were localized preferentially to R-positive bands throughout chromosome 9. Although the number of clones was roughly proportional to the R-band size, many more clones (ca. 80) were mapped at the q34 region compared to its physical size. Based on these cytogenetic mapping data, one can establish physical contig maps with cosmids and yeast artificial chromosomes and a genetic linkage map essential for positional cloning of responsible genes. PMID:8188268

Takahashi, E; Koyama, K; Hitomi, A; Itoh, H; Nakamura, Y

1994-01-15

270

Chromosomal translocation t(X;18) in human synovial sarcomas analyzed by fluorescence in situ hybridization using paraffin-embedded tissue.  

PubMed Central

Synovial sarcoma is characterized cytogenetically by translocation t(X;18)(p11.2;q11.2). In this study, 28 cases that had been diagnosed initially as synovial sarcoma, including 2 fibrosarcomas, and 1 leiomyosarcoma were collected and examined for translocation t(X;18) on paraffin-embedded tissues by fluorescence in situ hybridization (FISH). Of the synovial sarcomas, 25 showed findings consistent with translocation t(X;18) with an additional copy signal for the total probe of X and 18 chromosomes. The other three cases, as well as the two fibrosarcomas and the leiomyosarcoma, did not show this translocation. One (case 26) of three negative cases was diagnosed finally as leiomyosarcoma and another (case 27) as malignant peripheral nerve sheath tumor from histological and immunohistochemical analysis. Thus, in all, 25 (96%) of 26 synovial sarcomas showed findings consistent with translocation t(X;18). In summary, translocation t(X;18) is a chromosomal aberration specific for synovial sarcoma. The fluorescence in situ hybridization technique can be used even on cells from paraffin-embedded tissues, and is a useful diagnostic aid for synovial sarcoma. Images Figure 1 Figure 2

Nagao, K.; Ito, H.; Yoshida, H.

1996-01-01

271

Development of a fluorescence in situ hybridization protocol for the identification of micro-organisms associated with wastewater particles and flocs.  

PubMed

Fluorescence in situ hybridization (FISH) provides a unique tool to study micro-organisms associated with particles and flocs. FISH enables visual examination of micro-organisms while they are structurally intact and associated with particles. However, application of FISH to wastewater and sludge samples presents a specific set of problems. Wastewater samples generate high background fluorescence due to their organic and inorganic content making it difficult to differentiate a probe-conferred signal from naturally fluorescing particles with reasonable certainty. Furthermore, some of the FISH steps involve harsh treatment of samples, and are likely to disrupt the floc structure. This study developed a FISH protocol for studying micro-organisms that are associated with particles and flocs. The results indicate that choice of a proper fluorochrome and labeling technique is a key step in reducing the background fluorescence and non-specific binding, and increasing the intensity of the probe signal. Compared to other fluorochromes tested, CY3 worked very well and enabled the observation of particles and debris in red and probe signal from microbes in yellow. Fixation, hybridization, and washing steps disturbed the floc structure and particle-microbe association. Modifications to these steps were necessary, and were achieved by replacing centrifugation with filtration and employment of nylon filters. Microscope slides generated excellent quality images, but polycarbonate membrane filters performed better in preserving the floc structure. PMID:18821232

Ormeci, Banu; Linden, Karl G

2008-11-01

272

Bioconjugation of gold nanoparticles with DNA for in situ hybridization  

Microsoft Academic Search

This paper examines the in situ hybridization (ISH) coupled with nanoparticle bioconjugates in order to address the disadvantages of using fluorescently labeled probes for ISH, as well as to access the permeability of nanoparticles across bacterial cell walls. Accordingly, relatively high hybridization temperatures are used, resulting in nanogold precipitation and leaving dark precipitated granules under the microscope. In particular, high

Am Jang; Rameshwar Tatavarty; In S. Kim

2012-01-01

273

Delineation by fluorescence in situ hybridization of a single hemizygous chromosomal region associated with aposporous embryo sac formation in Pennisetum squamulatum and Cenchrus ciliaris.  

PubMed Central

Apomixis is a means of asexual reproduction by which plants produce embryos without meiosis and fertilization; thus the embryo is of clonal, maternal origin. We previously reported molecular markers showing no recombination with the trait for aposporous embryo sac development in Pennisetum squamulatum and Cenchrus ciliaris, and the collective single-dose alleles defined an apospory-specific genomic region (ASGR). Fluorescence in situ hybridization (FISH) was used to confirm that the ASGR is a hemizygous genomic region and to determine its chromosomal position with respect to rDNA loci and centromere repeats. We also documented chromosome transmission from P. squamulatum in several backcrosses (BCs) with P. glaucum using genomic in situ hybridization (GISH). One to three complete P. squamulatum chromosomes were detected in BC(6), but only one of the three hybridized with the ASGR-linked markers. In P. squamulatum and in all BCs examined, the apospory-linked markers were located in the distal region of the short arm of a single chromosome. All alien chromosomes behaved as univalents during meiosis and segregated randomly in BC(3) and later BC generations, but presence of the ASGR-carrier chromosome alone was sufficient to confer apospory. FISH results support our hypotheses that hemizygosity, proximity to centromeric sequences, and chromosome structure may all play a role in low recombination in the ASGR.

Goel, Shailendra; Chen, Zhenbang; Conner, Joann A; Akiyama, Yukio; Hanna, Wayne W; Ozias-Akins, Peggy

2003-01-01

274

A high-resolution cytogenetic map of human chromosome 5: Localization of 206 new cosmid markers by direct R-banding fluorescence in situ hybridization  

SciTech Connect

The authors have constructed a high-resolution cytogenetic map of human chromosome 5 with 206 new cosmid clones by direct R-banding fluorescence in situ hybridization. The fluorescent signals of 206 clones were evenly distributed throughout chromosome 5, although they were sublocalized preferentially to R-positive bands. This high-resolution cytogenetic map with an average distance of nearly 1 Mb will serve as an important resource for construction of a genetic linkage map, which is essential for positional cloning of responsible genes. Moreover, these mapping data provide many useful landmarks for the construction of contig maps of chromosome 5, as required in the Human Genome Project. 15 refs., 2 figs., 1 tab.

Takahashi, Ei-Ichi; Hitomi, Akitsu (National Institute of Radiological Sciences, Chiba (Japan)); Nakamura, Yusuke (Cancer Institute, Tokyo (Japan))

1993-07-01

275

Fluorescence in situ hybridization to interphase cell nuclei in suspension allows flow cytometric analysis of chromosome content and microscopic analysis of nuclear organization  

Microsoft Academic Search

Fluorescence hybridization to interphase nuclei in liquid suspension allows quantification of chromosome-specific DNA sequences using flow cytometry and the analysis of the three-dimensional positions of these sequences in the nucleus using fluorescence microscopy. The three-dimensional structure of nuclei is substantially intact after fluorescence hybridization in suspension, permitting the study of nuclear organization by optical sectioning. Images of the distribution of

Barbara Trask; Ger van den Engh; Dan Pinkel; Jim Mullikin; Fred Waldman; Herman van Dekken; Joe Gray

1988-01-01

276

A high-resolution cytogenetic map of human chromosome 3: localization of 291 new cosmid markers by direct R-banding fluorescence in situ hybridization.  

PubMed

We localized 291 new cosmid markers (including 65 RFLPs) on human chromosome 3 by direct R-banding fluorescence in situ hybridization. This system, which is based on fluorescence in situ hybridization combined with replicated prometaphase R-bands, allows the direct visualization of signals on R-banded prometaphases stained with propidium iodide and provides a more rapid and efficient method for genome mapping of cosmid clones. The signals of 291 markers examined here were localized preferentially to R-positive bands throughout chromosome 3. The detailed map positions of 366 clones and the characterization of 142 RFLPs, including the preliminary data reported by Yamakawa et al. (1991, Genomics 9: 536-543; and 11: 565-572), are summarized. This high-resolution cytogenetic map (average distance of 0.58 Mb), in conjunction with a genetic linkage map, can facilitate the analysis of chromosomal and molecular aberrations in genetic diseases and cancers. Furthermore, these mapping data will provide many useful landmarks for the construction of contig maps of chromosome 3. PMID:1354637

Takahashi, E; Yamakawa, K; Nakamura, Y; Hori, T

1992-08-01

277

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

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.

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

1999-01-01

278

Human gametes and zygotes studied by nonradioactive in situ hybridization  

Microsoft Academic Search

A nonradioactive in situ hybridization technique was applied to human gametes and abnormally fertilized or developed zygotes. Using haptenized chromosome-specific probes, visualization was obtained using immunocytochemistry to achieve a fluorescent stain on specific hybrids. Using a chromosome 1-specific DNA probe, almost all spermatozoa gave a positive result, i.e., one hybridization signal per cell could be observed. Furthermore, it was possible

M. H. E. C. Pieters; J. P. M. Geraedts; H. Meyer; J. C. M. Dumoulin; J. L. H. Evers; R. J. E. Jongbloed; P. M. Nederlof; S. van der Flier

1990-01-01

279

Detection of WWE2-related Lentisphaerae by 16S rRNA gene sequencing and fluorescence in situ hybridization in landfill leachate.  

PubMed

We collected samples of anaerobic landfill leachate from municipal solid waste landfill (Vert-le-Grand, France) and constructed 16S rRNA clone libraries using primers targeting Planctomycetes and relatives (Pla46F and 1390R). Analyses of 16S rRNA gene sequences resulted in the abundant representation of WWE2-related Lentisphaerae, members of the phylum Lentisphaerae, in the clone library (98% of the retrieved sequences). Although the sequences that are phylogenetically affiliated with the cultured isolate Victivallis vadensis were identified (WWE2 subgroup II), the majority of the sequences were affiliated with an uncultured Lentisphaerae lineage (WWE2 subgroup I). We designed oligonucleotides probes targeting the specific 16S rRNA gene regions of those 2 subgroups. Fluorescence in situ hybridization confirmed the abundance of the uncultivated WWE2 subgroup I in our leachate samples. PMID:20962908

Limam, Rim Driss; Bouchez, Théodore; Chouari, Rakia; Li, Tianlun; Barkallah, Insaf; Landoulsi, Ahmed; Sghir, Abdelghani

2010-10-01

280

Comparative mapping of DNA probes derived from the V{sub k} immunoglobulin gene regions on human and great ape chromosomes by fluorescence in situ hybridization  

SciTech Connect

Fluorescence in situ hybridization (FISH) of cosmid clones of human V{sub K} gene regions to human and primate chromosomes contributed to the dating of chromosome reorganizations in evolution. A clone from the K locus at 2p11-p12 (cos 106) hybridized to the assumed homologous chromosome bands in the chimpanzees Pan troglodytes (PTR) and P. paniscus (PPA), the Gorilla gorilla (GGO), and the orangutan Pongo Pygmaeus (PPY). Human and both chimpanzees differed from gorilla and orangutan by the mapping of cos 170, a clone derived from chromosome 2cen-q11.2; the transposition of this orphon to the other side of the centromere can, therefore, be dated after the human/chimpanzee and gorilla divergence. Hybridization to homologous bands was also found with a cosmid clone containing a V{sub K}I orphon located on chromosome 1 (cos 115, main signal at 1q31-q32), although the probe is not fully unique. Also, a clone derived from the orphon V{sub K} region on chromosome 22q11 (cos 121) hybridized to the homologous bands in the great apes. This indicates that the orphons on human chromosomes 1 and 22 had been translocated early in primate evolution. 18 refs., 2 figs.

Arnold, N.; Wienberg, J.; Ermert, K. [Universitaet Muenchen (Germany)] [and others] [Universitaet Muenchen (Germany); and others

1995-03-01

281

[Determination of HER2 by fluorescence in situ hybridization (FISH) in breast cancer. Experience of the Reference Laboratory of Lisbon].  

PubMed

The knowledgement of HER2/c-erbB-2 status in breast cancer is essential for the eligibility of the patients for therapy with monoclonal antibody anti-HER-2/trastuzumab. From the various existent techniques for the determination of HER-2, the most widely used are: immunohistochemistry (IHC), that estimates the protein expression, and in situ hybridization (FISH), that evaluates its amplification. FISH is essential as complementary of IHQ in tumors with equivocal or non interpreted staining, and also in those with complete weak to moderate membrane staining in >10 % of neoplastic cells (score 2+) because, of these, only cases with gene amplification (FISH positive) respond to therapy. Recent studies also support that FISH should be performed in cases with score 3+, because 10-12% of them don't have amplification. The expensive equipment and reagents for standard FISH method and the need of extensive training and expertise for both the technique and the evaluation, justify a centralized testing in Reference Laboratories. Our aim was to describe the work of the Department of Pathology of the Portuguese Institute of Oncology of Lisbon as a Reference Laboratory in the execution of FISH technique and to present the results obtained from May 2001 to August 2004. FISH was performed with Kit INFORM HER2/neu Gene Detection System, using the BenckMark system of Ventana. We evaluated a series of 4499 invasive carcinoma primary of the breast, that included 587 cases of the Portuguese Institute of Oncology of Lisboa and 3912 cases from different Hospitals of Lisboa, Cascais, Almada, Barreiro, Santarém, Evora, Faro, Portimăo, Guimarăes, Funchal and Angra do Heroísmo. We verified that 591 cases (13.5%) had HER2 gene amplification, being those patients eligible for therapy with trastuzumab. PMID:16684481

André, Saudade; Tomás, Ana Raquel; Fonseca, Ricardo

2005-01-01

282

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

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.

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

1995-09-11

283

Granulocytic sarcoma presenting as pneumonia in a child with t(8;21) acute myelogenous leukemia: diagnosis by fluorescent in situ hybridization.  

PubMed

Granulocytic sarcoma is a soft tissue collection of leukemic cells. The authors describe a 4-year-old boy with M2 acute myelogenous leukemia (AML) who presented with fever, mild nonproductive cough, and hematemesis. Although he was initially diagnosed with nodular pneumonia, rapid resolution of a pulmonary infiltrate following induction chemotherapy was suggestive of a pulmonary granulocytic sarcoma. Interphase fluorescent in situ hybridization (FISH) of the lung biopsy specimen for the t(8;21)(q22;q22) translocation confirmed the retrospective diagnosis of a well-differentiated pulmonary granulocytic sarcoma. Pulmonary granulocytic sarcomas may be underrecognized in children with AML; this may delay anti-leukemic therapy and may lead to ineffective therapy if misdiagnosed as pneumonia. PMID:15218417

Lee, Dean A; Harris, Charles P; Gresik, Vicky M; Rao, Pulivarthi; Lau, Ching C

2004-07-01

284

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)

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.

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

1998-01-01

285

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

SciTech Connect

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.

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

1994-11-15

286

A methodology to infer gene networks from spatial patterns of expression - An application to fluorescence in situ hybridization images  

PubMed Central

The proper functional development of a multicellular organism depends on an intricate network of interacting genes that are expressed in accurate temporal and spatial patterns across different tissues. Complex inhibitory and excitatory interactions among genes control the territorial differences that explain specialized cell fates, embryo polarization and tissues architecture in metazoan. Given the nature of the regulatory gene networks, similarity of expression patterns can identify genes and with similar roles. The inference and analysis of the gene interaction networks through complex networks tools can reveal important aspects of the biological system modeled. Here we suggest an image analysis pipeline to quantify co-localization patterns in in situ hybridization images of Drosophila embryos and, based on these patterns, infer gene networks. We analyze the spatial dispersion of the gene expression and show the gene interaction networks for different developmental stages. Our results suggest that the inference of developmental networks based on spatial expression data are biologically relevant and represents a potential tool for the understanding of animal development.

Campiteli, Monica Guimaraes; Comin, Cesar Henrique; Costa, Luciano da Fontoura; Babu, M Madan; Cesar, Roberto Marcondes

2014-01-01

287

Differentiation of Methanosaeta concilii and Methanosarcina barkeri in Anaerobic Mesophilic Granular Sludge by Fluorescent In Situ Hybridization and Confocal Scanning Laser Microscopy†  

PubMed Central

Oligonucleotide probes, designed from genes coding for 16S rRNA, were developed to differentiate Methanosaeta concilii, Methanosarcina barkeri, and mesophilic methanogens. All M. concilii oligonucleotide probes (designated MS1, MS2, and MS5) hybridized specifically with the target DNA, but MS5 was the most specific M. concilii oligonucleotide probe. Methanosarcina barkeri oligonucleotide probes (designated MB1, MB3, and MB4) hybridized with different Methanosarcina species. The MB4 probe specifically detected Methanosarcina barkeri, and the MB3 probe detected the presence of all mesophilic Methanosarcina species. These new oligonucleotide probes facilitated the identification, localization, and quantification of the specific relative abundance of M. concilii and Methanosarcina barkeri, which play important roles in methanogenesis. The combined use of fluorescent in situ hybridization with confocal scanning laser microscopy demonstrated that anaerobic granule topography depends on granule origin and feeding. Protein-fed granules showed no layered structure with a random distribution of M. concilii. In contrast, a layered structure developed in methanol-enriched granules, where M. barkeri growth was induced in an outer layer. This outer layer was followed by a layer composed of M. concilii, with an inner core of M. concilii and other bacteria.

Rocheleau, Sylvie; Greer, Charles W.; Lawrence, John R.; Cantin, Christiane; Laramee, Louise; Guiot, Serge R.

1999-01-01

288

Detection of Ralstonia solanacearum, Which Causes Brown Rot of Potato, by Fluorescent In Situ Hybridization with 23S rRNA-Targeted Probes  

PubMed Central

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.

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

1998-01-01

289

Diagnostic Examination of Human Intestinal Spirochetosis by Fluorescent In Situ Hybridization for Brachyspira aalborgi, Brachyspira pilosicoli, and Other Species of the Genus Brachyspira (Serpulina)  

PubMed Central

Human intestinal spirochetosis, characterized by end-on attachment of densely packed spirochetes to the epithelial surface of the large intestines as a fringe has been associated with the weakly beta-hemolytic spirochetes Brachyspira aalborgi and Brachyspira (Serpulina) pilosicoli. In this study, fluorescent in situ hybridization with oligonucleotide probes targeting 16S or 23S rRNA of B. aalborgi, B. pilosicoli, and the genus Brachyspira was applied to 40 sections of formalin-fixed, paraffin-embedded intestinal biopsy specimens from 23 Danish and 15 Norwegian patients with histologic evidence of intestinal spirochetosis. Five biopsy specimens from patients without intestinal spirochetosis and three samples from pigs with experimental B. pilosicoli colitis were examined as well. In addition, the 16S ribosomal DNAs of two clinical isolates of B. aalborgi were sequenced, and a PCR procedure was developed for the identification of B. aalborgi in cultures. The genotypic characteristics of the two clinical isolates showed very high (99.5%) similarity with two existing isolates, the type strain of B. aalborgi and a Swedish isolate. Hybridization with the Brachyspira genus-specific probe revealed a brightly fluorescing fringe of spirochetes on the epithelia of 39 biopsy specimens, whereas 1 biopsy specimen was hybridization negative. The spirochetes in biopsy specimens from 13 Danish and 8 Norwegian patients (55.3%) were identified as B. aalborgi. The spirochetes in the biopsy specimens from the other 17 patients hybridized only with the Brachyspira probe, possibly demonstrating the involvement of as-yet-uncharacterized Brachyspira spirochetes in human intestinal spirochetosis.

Jensen, T. K.; Boye, M.; Ahrens, P.; Korsager, B.; Teglbjaerg, P. S.; Lindboe, C. F.; M?ller, K.

2001-01-01

290

Specific Detection of Pasteurella multocida in Chickens with Fowl Cholera and in Pig Lung Tissues Using Fluorescent rRNA In Situ Hybridization  

PubMed Central

A Pasteurella multocida species-specific oligonucleotide probe, pmhyb449, targeting 16S rRNA was designed and evaluated by whole-cell hybridization against 22 selected reference strains in animal tissues. It differentiated P. multocida from other bacterial species of the families Pasteurellaceae and Enterobacteriaceae and also from divergent species of the order Cytophagales (except biovar 2 strains of Pasteurella avium and Pasteurella canis, which have high 16S rRNA similarity to P. multocida). The potential of the probe for specific identification and differentiation of P. multocida was further detected in formalin-fixed paraffin-embedded lung tissues from experimental fowl cholera in chickens and infections in pigs. In chicken lung tissues P. multocida cells were detected singly, in pairs, as microcolonies, and as massive colonies within air capillaries (septa and lumen), parabronchial septa, and blood vessels (wall and lumen). In pig lung, postmortem-injected P. multocida was detected in the alveoli (lumen and wall), and in both animals the bacterial cells were seen in the bronchi. The results showed that with the oligonucleotide probe pmhyb449, fluorescent in situ hybridization is a suitable and fast method for specific detection of P. multocida in histological formalin-fixed tissues. The test was replicable and reproducible and is recommended as a supplementary test for diagnosis and as a tool in pathogenesis studies of fowl cholera and respiratory tract infections in pigs due to P. multocida.

Mbuthia, Paul Gichohi; Christensen, Henrik; Boye, Mette; Petersen, Kamille Majken Dumong; Bisgaard, Magne; Nyaga, Phillip Njeru; Olsen, John Elmerdahl

2001-01-01

291

Mapping of the human thromboxane synthase gene (TBXAS1) to chromosome 7q34-q35 by two-color fluorescence in situ hybridization  

SciTech Connect

Thromboxane synthase (TS) catalyzes the conversion of the prostaglandin endoperoxide into thromboxane A[sub 2] (TxA[sub 2]), a potent vasoconstrictor and inducer of platelet aggregation. In concert with prostacyclin, TxA[sub 2] plays a pivotal role in the maintenance of hemostasis. Deficiency of platelet TS activity has been shown to result in bleeding disorders. The potent effect of TxA[sub 2] on platelet function and vascular activity suggests a possible involvement of TS in normal and pathophysiological conditions such as cardiovascular disease. To aid in establishing the correlation of TS to disease states, the authors localized the human TS gene (TBXAS1) to chromosome 7q34-q35 using dual-color fluorescence in situ hybridization. 21 refs., 2 figs.

Chase, M.B.; Baek, Seung Joon; Purtell, D.C.; Schwartz, S. (Univ. of Maryland Medical School, Baltimore, MD (United States)); Shen, Rong Fong (Univ. of Maryland Medical School, Baltimore, MD (United States) Maryland Biotechnology Institute, Baltimore, MD (United States))

1993-06-01

292

Comparative Fluorescence in Situ Hybridization Mapping of a 431-kb Arabidopsis thaliana Bacterial Artificial Chromosome Contig Reveals the Role of Chromosomal Duplications in the Expansion of the Brassica rapa Genome  

Microsoft Academic Search

Comparative genome studies are important contributors to our understanding of genome evolution. Most comparative genome studies in plants have been based on genetic mapping of homologous DNA loci in different genomes. Large-scale comparative physical mapping has been hindered by the lack of efficient and affordable techniques. We report here the adaptation of fluorescence in situ hybridization (FISH) techniques for comparative

Scott A. Jackson; Zhukuan Cheng; Ming Li Wang; Howard M. Goodman; Jiming Jiang

2000-01-01

293

Mosaic vs. nonmosaic trisomy 9: Report of a liveborn infant evaluated by fluorescence in situ hybridization and review of the literature  

SciTech Connect

We report on a newborn infant with multiple congenital anomalies and apparent nonmosaic trisomy 9 in the blood (by conventional cytogenetic studies) who died shortly after birth. Clinical observations at birth and autopsy are compared with phenotypes of mosaic and nonmosaic trisomy 9 cases reported previously. Unlike the initial cytogenetic analysis, fluorescence in situ hybridization (FISH) studies of metaphase and interphase blood cells and skin fibroblasts detected the presence of euploid and trisomy 9 cells. These results suggest that earlier reports of trisomy 9, which relied on conventional chromosome analysis of a few metaphase cells and/or only one tissue type, may not have excluded mosaicism, and that trisomy 9 may be viable only in the mosaic state. 39 refs., 3 figs., 2 tabs.

Cantu, E.S.; Eicher, D.J.; Shashidhar Pai, G.; Donahue, C.J.; Harley, R.A. [Medical Univ. of South Carolina, Chalreston, SC (United States)

1996-04-24

294

Reliable protocols for whole-mount fluorescent in situ hybridization (FISH) in the pea aphid Acyrthosiphon pisum: a comprehensive survey and analysis.  

PubMed

RNA in situ hybridization (ISH), including chromogenic ISH (CISH) and fluorescent ISH (FISH), has become a powerful tool for revealing the spatial distribution of gene transcripts in model organisms. Previously, we developed a robust protocol for whole-mount RNA CISH in the pea aphid Acyrthosiphon pisum, an emerging insect genomic model. In order to improve the resolving capacity of gene detection, we comprehensively surveyed current protocols of whole-mount RNA-FISH and developed protocols that allow, using confocal microscopy, clearer visualization of target messenger RNAs (mRNAs) - including those subcellularly localized and those with spatially overlapping expression. We find that Fast dye-based substrate fluorescence (SF), tyramide signal amplification (TSA), and TSA Plus all enable identifying gene expression thanks to multiplex amplification of fluorescent signals. By contrast, methods of direct fluorescence (DF) do not allow visualizing signals. Detection of a single gene target was achieved with SF and TSA Plus for most mRNAs, whereas TSA only allowed visualization of abundant transcripts such as Apvas1 and Appiwi2 in the germ cells. For detection of multiple gene targets using double FISH, we recommend: (i) TSA/TSA, rather than TSA Plus/TSA Plus for colocalized mRNAs abundantly expressed in germ cells, as proteinase K treatment can be omitted; and (ii) SF/TSA Plus for other gene targets such as Apen1 and Apen2 as inactivation of enzyme conjugates is not required. SF/SF is not ideal for double FISH experiments due to signal blurring. Based on these new conditions for RNA-FISH, we have obtained a better understanding of germline specification and embryonic segmentation in the pea aphid. We anticipate that the RNA-FISH protocols for the pea aphid may also be used for other aphids and possibly other insect species, thus expanding the range of species from which useful insights into development and evolution may be obtained. PMID:24850784

Chung, Chen-yo; Cook, Charles E; Lin, Gee-way; Huang, Ting-Yu; Chang, Chun-che

2014-06-01

295

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

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.

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

296

5-bp Classical Satellite DNA Loci from Chromosome-1 Instability in Cervical Neoplasia Detected by DNA Breakage Detection/Fluorescence in Situ Hybridization (DBD-FISH)  

PubMed Central

We aimed to evaluate the association between the progressive stages of cervical neoplasia and DNA damage in 5-bp classical satellite DNA sequences from chromosome-1 in cervical epithelium and in peripheral blood lymphocytes using DNA breakage detection/fluorescence in situ hybridization (DBD-FISH). A hospital-based unmatched case-control study was conducted in 2011 with a sample of 30 women grouped according to disease stage and selected according to histological diagnosis; 10 with low-grade squamous intraepithelial lesions (LG-SIL), 10 with high-grade SIL (HG-SIL), and 10 with no cervical lesions, from the Unidad Medica de Alta Especialidad of The Mexican Social Security Institute, IMSS, Mexico. Specific chromosome damage levels in 5-bp classical satellite DNA sequences from chromosome-1 were evaluated in cervical epithelium and peripheral blood lymphocytes using the DBD-FISH technique. Whole-genome DNA hybridization was used as a reference for the level of damage. Results of Kruskal-Wallis test showed a significant increase according to neoplastic development in both tissues. The instability of 5-bp classical satellite DNA sequences from chromosome-1 was evidenced using chromosome-orientation FISH. In conclusion, we suggest that the progression to malignant transformation involves an increase in the instability of 5-bp classical satellite DNA sequences from chromosome-1.

Cortes-Gutierrez, Elva I.; Ortiz-Hernandez, Brenda L.; Davila-Rodriguez, Martha I.; Cerda-Flores, Ricardo M; Fernandez, Jose Luis; Lopez-Fernandez, Carmen; Gosalvez, Jaime

2013-01-01

297

Enumeration of Vibrio parahaemolyticus in the viable but nonculturable state using direct plate counts and recognition of individual gene fluorescence in situ hybridization.  

PubMed

Vibrio parahaemolyticus is a gram-negative, halophilic bacterium indigenous to marine and estuarine environments and it is capable of causing food and water-borne illness in humans. It can also cause disease in marine animals, including cultured species. Currently, culture-based techniques are used for quantification of V. parahaemolyticus in environmental samples; however, these can be misleading as they fail to detect V. parahaemolyticus in a viable but nonculturable (VBNC) state which leads to an underestimation of the population density. In this study, we used a novel fluorescence visualization technique, called recognition of individual gene fluorescence in situ hybridization (RING-FISH), which targets chromosomal DNA for enumeration. A polynucleotide probe labeled with Cyanine 3 (Cy3) was created corresponding to the ubiquitous V. parahaemolyticus gene that codes for thermolabile hemolysin (tlh). When coupled with the Kogure method to distinguish viable from dead cells, RING-FISH probes reliably enumerated total, viable V. parahaemolyticus. The probe was tested for sensitivity and specificity against a pure culture of tlh(+), tdh(-), trh(-)V. parahaemolyticus, pure cultures of Vibrio vulnificus, Vibrio harveyi, Vibrio alginolyticus and Vibrio fischeri, and a mixed environmental sample. This research will provide additional tools for a better understanding of the risk these environmental organisms pose to human health. PMID:21329738

Griffitt, Kimberly J; Noriea, Nicholas F; Johnson, Crystal N; Grimes, D Jay

2011-05-01

298

Simultaneous 16S and 18S rRNA fluorescence in situ hybridization (FISH) on LR White sections demonstrated in Vestimentifera (Siboglinidae) tubeworms.  

PubMed

Traditional morphological identification of invertebrate marine species is limited in early life history stages for many taxa. In this study, we demonstrate, by example of Vestimentiferan tubeworms (Siboglinidae, Polychaeta), that the simultaneous fluorescence in situ hybridization (FISH) of both eukaryotic host and bacterial symbiont cells is possible on a single semi-thin (1 ?m) section. This allows the identification of host specimens to species level as well as offering visualization of bacteria distributed within the host tissue. Previously published 18S rRNA host-specific oligonucleotide probes for Riftia pachyptila, Tevnia jerichonana and a newly designed Oasisia alvinae probe, as well as a 16S rRNA probe targeting symbionts found in all host species, were applied. A number of standard fixation and hybridization parameters were tested and optimized for the best possible signal intensity and cellular resolution. Ethanol conserved samples embedded in LR White low viscosity resin yielded the best results with regard to both signal intensity and resolution. We show that extended storage times of specimens does not affect the quality of signals attained by FISH and use our protocol to identify morphologically unidentifiable tubeworm individuals from a small data set, conforming to previous findings in succession studies of the Siboglinidae family. PMID:21507466

Schimak, Mario P; Toenshoff, Elena R; Bright, Monika

2012-02-01

299

Chromosomal composition of micronuclei in mouse bone marrow treated with rifampicin and nicotine, analyzed by multicolor fluorescence in situ hybridization with pancentromeric DNA probe.  

PubMed

The mechanism underlying the induction of micronuclei induced by rifampicin and nicotine in mouse bone marrow was investigated by fluorescence in situ hybridization assay using mouse minor satellite DNA probe. Colchicine and mitomycin, known to be predominantly clastogenic and aneugenic, respectively were used as positive controls and these compounds produced the expected responses. In animals treated with different doses of rifampicin (10-320 mg/kg), the frequencies of micronucleated polychromatic erythrocytes (MNPCE) increased significantly after treatment with 160 and 320 mg/kg. Furthermore, rifampicin caused a significant depression of erythroblast proliferation at the high dose. At the two highest doses of 160 and 320 mg/kg rifampicin, a total of 0.96% and 1.44% MNPCE, respectively were found. Of the rifampicin-induced signal-positive MNPCE, an average of 58.1% of them was centromere-negative, reflecting the clastogenic activity of rifampicin. Correspondingly, about 41.9% of induced MNPCE were centromere-positive, representing the aneugenic activity of rifampicin. Eight and 16 mg/kg of nicotine induced 0.84% and 1.2% MNPCE, respectively, and of these an average of 29.5% showed one or more fluorescent signals, reflecting the predominant clastogenic activity of nicotine. The results obtained demonstrate that rifampicin induced both chromosome breakage and numerical chromosomal abnormalities, whereas nicotine induced one type of MNPCE and it could be considered a clastogenic agent. PMID:17420080

Attia, Sabry M

2007-06-01

300

mRNA-targeted Fluorescent in Situ Hybridization (FISH) of Gram-negative Bacteria Without Template Amplification or Tyramide Signal Amplification  

SciTech Connect

Technologies are needed to study gene expression at the level of individual cells within a population or microbial community. Fluorescent in situ hybridization (FISH) supplies high-resolution spatial information and has been widely applied to study microbial communities at the rRNA level. While mRNA-targeted FISH has been popular for studying gene expression in eukaryotic cells, very little success has been achieved with prokaryotes. At present, detection of specific mRNAs in individual prokaryotic cells requires the use of in situ-RT-PCR or tyramide signal amplification (TSA). In this study we used DNA oligonucleotide probes labeled with a single near-infrared dye in FISH assays to detect multicopy plasmid-based and endogenous mRNA molecules in Escherichia coli and Shewanella oneidensis MR-1. We took advantage of the fact there is much less background signal produced by biological materials and support matrices in the near-infrared spectrum and thus long camera exposure times could be used. In addition, we demonstrate that a combination of probes targeting both rRNA and mRNA could be successfully employed within the same FISH assay. These results, as well as ongoing R&D improvements in NIR and infrared dyes, indicate the FISH approach we demonstrated could be applied in certain environmental settings to monitor gene expression in mixed populations.

Coleman, James R.; Culley, David E.; Chrisler, William B.; Brockman, Fred J.

2007-12-01

301

Etoposide and merbarone are clastogenic and aneugenic in the mouse bone marrow micronucleus test complemented by fluorescence in situ hybridization with the mouse minor satellite DNA probe.  

PubMed

The topoisomerase II (topo II) inhibitors etoposide (VP-16) and merbarone (MER) were investigated with the in vivo micronucleus test (MN test) combined with fluorescence in situ hybridization (FISH) using the mouse minor satellite DNA probe to discriminate MN of clastogenic and aneugenic origin. All experiments were performed with male (102/ElxC3H/El) F1 mice bred in the mouse colony of the GSF Research Center. The sample size per experimental group was five animals and 2,000 polychromatic erythrocytes (PCE) were scored per animal from coded slides in the conventional MN test. A separate set of coded slides was used for the FISH analysis. All treatments consisted of single intraperitoneal injections. Colchicine (COL, 3 mg/kg) and mitomycin (MMC, 1 mg/kg) were used as a positive control aneugen and clastogen, respectively, and these compounds produced the expected responses. A dose of 1 mg/kg VP-16 induced 3.44% MNPCE (compared to the concurrent solvent control of 0.37%, P < 0.001) and of these 39.9% (1.4% MNPCE) showed one or more fluorescent signals. MER (7.5-60 mg/kg) increased the MNPCE frequencies in a dose-dependent manner, with 15 mg/kg being the lowest positive dose. At the highest dose of 60 mg/kg of MER, a total of 4.26% MNPCE were found (compared to 0.31% in the concurrent solvent control, P < 0.001) and of these 46.2% (2.0% MNPCE) contained one or more fluorescent signals. The data demonstrate that VP-16 and MER induced both clastogenic and aneugenic events despite their different modes of topo II inhibition. PMID:12605378

Attia, S M; Kliesch, U; Schriever-Schwemmer, G; Badary, O A; Hamada, F M; Adler, I-D

2003-01-01

302

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

SciTech Connect

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.

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

303

Chromosome 11 aneusomy in esophageal cancers and precancerous lesions- an early event in neoplastic transformation: An interphase fluorescence in situ hybridization study from south India  

PubMed Central

AIM: To detect aneusomic changes with respect to chromosome 11 copy number in esophageal precancers and cancers wherein the generation of cancer-specific phenotypes is believed to be associated with specific chromosomal aneuploidies. METHODS: We performed fluorescence in situ hybridization (FISH) on esophageal tissue paraffin sections to analyze changes in chromosome 11 copy number using apotome-generated images by optical sectioning microscopy. Sections were prepared from esophageal tumor tissue, tissues showing preneoplastic changes and histologically normal tissues (control) obtained from patients referred to the clinic for endoscopic evaluation. RESULTS: Our results demonstrated that aneusomy was seen in all the cancers and preneoplastic tissues, while none of the controls showed aneusomic cells. There was no increase in aneusomy from precancers to cancers. CONCLUSION: Our results suggest that evaluation of chromosome 11 aneusomy in esophageal tissue using FISH with an appropriate signal capture-analysis system, can be used as an ancillary molecular marker predictive of early neoplastic changes. Future studies can be directed towards the genes on chromosome 11, which may play a role in the neoplastic transformation of esophageal precancerous lesions to cancers.

Mohan, Vasavi; Ponnala, Shivani; Reddy, Hemakumar M; Sistla, Radha; Jesudasan, Rachel A; Ahuja, Yog Raj; Hasan, Qurratulain

2007-01-01

304

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

SciTech Connect

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.

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

1993-07-01

305

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

PubMed Central

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.

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

306

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

PubMed

We 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, we identified 71 polymorphic systems with 43 markers; 11 of these are VNTRs. As our 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. PMID:8104866

Inazawa, J; Saito, H; Ariyama, T; Abe, T; Nakamura, Y

1993-07-01

307

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

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

Yilmaz, Ozlem; Demiray, Ebru

2007-02-01

308

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

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.

Yilmaz, Ozlem; Demiray, Ebru

2007-01-01

309

Ewing sarcoma/peripheral primitive neuroectodermal tumor: adult abdominal tumors with an Ewing sarcoma gene rearrangement demonstrated by fluorescence in situ hybridization in paraffin sections.  

PubMed

The differential diagnosis of small round cell tumors is exhaustive and requires ancillary studies. Relatively recently, fluorescence in situ hybridization (FISH) using probes for specific gene rearrangements has gained wide acceptance. This technique is particularly useful in the differential diagnosis of Ewing sarcoma/primitive neuroectodermal tumor (ES/PNET) and desmoplastic small round-cell tumor (DSRCT). In ES/PNET, the EWS gene is juxtaposed to the FLI-1 gene in 85% of cases and to the ERG gene in another 7% of cases; the EWS gene is juxtaposed to the WTI gene in DSRCT. Documentation of the EWS gene rearrangements in EWS/PNET has previously been demonstrated in frozen tissue. We report 2 unusual cases of EWS/PNET diagnosed in abdominal tumors in adults. Although the immunohistochemical results supported a diagnosis of ES/PNET, 1 case morphologically resembled DSRCT. The diagnosis in these 2 cases was confirmed by the FISH demonstration of EWS/FLI-1 gene fusion in paraffin-embedded tissue. Thus, the usefulness of FISH demonstration of an EWS gene rearrangement with these specific probes in such unusual cases is supported and is demonstrated in paraffin-embedded tissue. PMID:15354743

Gardner, Laura J; Ayala, Alberto G; Monforte, Hector L; Dunphy, Cherie H

2004-06-01

310

Identification of Treponema pedis as the predominant Treponema species in porcine skin ulcers by fluorescence in situ hybridization and high-throughput sequencing.  

PubMed

Skin lesions often seen in pig production are of great animal welfare concern. To study the potential role of Treponema bacteria in porcine skin ulcers, we investigated the presence and distribution of these organisms in decubital shoulder ulcers (n=51) and ear necroses (n=54) by fluorescence in situ hybridization (FISH) and high-throughput sequencing. In addition, two cases of facial ulcers and five cases of other skin ulcers were included in the study. Samples from all 112 skin lesions and intact skin from pigs without skin ulcers (n=14) were screened by FISH. Three different oligonucleotide probes targeting 16S rRNA were used, specific for domain bacterium, Treponema spp. and species T. pedis. Screening showed that two cases each of facial and other ulcers, 35 (69%) of shoulder ulcers and 32 (59%) of ear necroses were positive for Treponema spp. T. pedis was the unequivocally, predominant species typically constituting more than 90% of the treponemes in a lesion, assessed visually by microscopy. Altogether, T. pedis was demonstrated in 69 of the 71 Treponema spp. positive lesions. We conclude that Treponema spp. are frequently present and abundant in various skin ulcers of pigs. The results from this study point toward an important role of T. pedis as a secondary bacterial infection in porcine skin ulcers, especially in severe and chronic lesions. PMID:24725449

Karlsson, Frida; Klitgaard, Kirstine; Jensen, Tim Kĺre

2014-06-25

311

Automated Design of Probes for rRNA-Targeted Fluorescence In Situ Hybridization Reveals the Advantages of Using Dual Probes for Accurate Identification.  

PubMed

Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu). PMID:24928876

Wright, Erik S; Yilmaz, L Safak; Corcoran, Andrew M; Okten, Hatice E; Noguera, Daniel R

2014-08-15

312

The genes for the {alpha}-type HC3 (PMSA2) and {beta}-type HC5 (PMSB1) subunits of human proteasomes map to chromosomes 6q27 and 7p12-p13 by fluorescence in situ hybridization  

SciTech Connect

The authors have determined the locations of the genes for the two subunits, HC3 and HC5, by fluorescence in situ hybridization (FISH). Chromosome spreads were obtained from phytohemagglutinin-stimulated blood lymphocytes of a healthy donor after thymidine synchronization and bromodeoxyuridine incorporation by the method of Takahashi et al. Genomic DNA fragments of HC3 (4.3 kb, including exons 3, 4, and 5) and HC5 (7.5 kb including exons 1 and 2) (11) were labeled with biotin-16-dUTP by nick-translation. In situ hybridization was performed according to Lichter et al. in the presence of COT-1 DNA as a competitor. Hybridized probe was detected with FITC-conjugated avidin without further signal amplification. Comparison of the fluorescence signals and the banding patterns of the chromosomes indicated that the HC3 and HC5 genes were located on chromosome band 6q27 and 7p12-p13, respectively.

Okumura, Katsuzumi; Nogami, Masahiro; Taguchi, Hiroshi [Mie Univ., Tsu (Japan)] [and others] [Mie Univ., Tsu (Japan); and others

1995-05-20

313

Double-color fluorescence in situ hybridization (FISH) for the detection of Bacillus anthracis spores in environmental samples with a novel permeabilization protocol.  

PubMed

For anti-bioterrorism measures against the use of Bacillus anthracis, a double-color fluorescence in situ hybridization (FISH) is proposed, for the rapid and specific detection of B. anthracis. The probes were designed based on the differences in 16S and 23S rRNA genes of B. cereus group. A new permeabilization protocol was developed to enhance the permeability of FISH probes into B. anthracis spores. The highest detection rate (90.8 ± 0.69) of B. anthracis spores by FISH was obtained with successive incubation steps with 50% ethanol at 80 °C, a mixture of SDS/DTT solution (10mg/ml SDS, 50mM DTT) at 65 °C and finally in a lysozyme solution (20mg/ml) at 37 °C for 30 min each. This protocol was evaluated for the detection of B. anthracis spores in soil and air samples after adding formalin-fixed spores at different densities. The results have proven the success of double-color FISH in detecting B. anthracis spores in air samples in the range of 10(3) spores/m(3) and above. Conversely, for detecting B. anthracis spores in a soil sample, the lowest detection limit was found to be 10(7) spores/g dry soils. These results confirm the applicability of the developed permeabilization protocol, combined with the double-color FISH technique in specific detection of B. anthracis in soil and air samples. PMID:23523967

Weerasekara, M L M A W; Ryuda, Noriko; Miyamoto, Hiroshi; Okumura, Toru; Ueno, Daisuke; Inoue, Koichi; Someya, Takashi

2013-06-01

314

Diversity of cultivable methane-oxidizing bacteria in microsites of a rice paddy field: investigation by cultivation method and fluorescence in situ hybridization (FISH).  

PubMed

The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH? emission from this ecosystem. PMID:22446309

Dianou, Dayéri; Ueno, Chihoko; Ogiso, Takuya; Kimura, Makoto; Asakawa, Susumu

2012-01-01

315

Diversity of Cultivable Methane-Oxidizing Bacteria in Microsites of a Rice Paddy Field: Investigation by Cultivation Method and Fluorescence in situ Hybridization (FISH)  

PubMed Central

The diversity of cultivable methane-oxidizing bacteria (MOB) in the rice paddy field ecosystem was investigated by combined culture-dependent and fluorescence in situ hybridization (FISH) techniques. Seven microsites of a Japanese rice paddy field were the focus of the study: floodwater, surface soil, bulk soil, rhizosphere soil, root, basal stem of rice plant, and rice stumps of previous harvest. Based on pmoA gene analysis and transmission electron microscopy (TEM), four type I, and nine type II MOB isolates were obtained from the highest dilution series of enrichment cultures. The type I MOB isolates included a novel species in the genus Methylomonas from floodwater and this is the first type I MOB strain isolated from floodwater of a rice paddy field. In the type I MOB, two isolates from stumps were closely related to Methylomonas spp.; one isolate obtained from rhizosphere soil was most related to Methyloccocus-Methylocaldum-Methylogaea clade. Almost all the type II MOB isolates were related to Methylocystis methanotrophs. FISH confirmed the presence of both types I and II MOB in all the microsites and in the related enrichment cultures. The study reported, for the first time, the diversity of cultivable methanotrophs including a novel species of type I MOB in rice paddy field compartments. Refining growth media and culture conditions, in combination with molecular approaches, will allow us to broaden our knowledge on the MOB community in the rice paddy field ecosystem and consequently to implement strategies for mitigating CH4 emission from this ecosystem.

Dianou, Dayeri; Ueno, Chihoko; Ogiso, Takuya; Kimura, Makoto; Asakawa, Susumu

2012-01-01

316

Anatomic site-specific patterns of gene copy number gains in skin, mucosal, and uveal melanomas detected by fluorescence in situ hybridization.  

PubMed

To assess the differences between melanomas of different location and different etiology, 372 malignant melanomas were brought in a tissue microarray format. The collection included 23 acral and 118 non-acral skin melanomas, 9 mucosal melanomas, 100 uveal melanomas, and 122 melanoma metastases. Fluorescence in situ hybridization (FISH) was used to assess copy number changes of the cyclin D1 (CCND1), MDM2, c-myc (MYC), and HER2 genes. FISH analysis revealed distinct differences between melanomas from different locations. CCND1 amplifications were detected in skin melanomas from sites with chronic sun exposure (6 of 32 cases), acral melanomas (4 of 17 cases), and mucosal melanomas (one of ten cases) but not in uveal melanomas. High-level MDM2 amplifications were exclusively present in acral melanomas (2 of 19 cases). MYC copy number gains were detected in 32 of 71 uveal melanomas, five of eight mucosal melanomas, and 6 of 67 melanomas from sites with intermittent sun exposure but not in acral melanomas nor melanomas from sites with chronic sun exposure. Alterations of the MYC gene were associated with advanced tumor stage. There were no high-level HER2 amplifications. Site-specific genetic and epigenetic features may impact the response of melanomas to various anti-cancer drugs and should be considered in future studies on the molecular pathogenesis of malignant melanomas. PMID:16523260

Glatz-Krieger, Katharina; Pache, Mona; Tapia, Coya; Fuchs, Alain; Savic, Spasenija; Glatz, Dieter; Mihatsch, Michael; Meyer, Peter

2006-09-01

317

Clonal evolution in chronic lymphocytic leukemia detected by fluorescence in situ hybridization and conventional cytogenetics after stimulation with CpG oligonucleotides and interleukin-2: a prospective analysis.  

PubMed

Chronic lymphocytic leukemia (CLL) patients may acquire new chromosome abnormalities during the course of their disease. Clonal evolution (CE) has been detected by conventional chromosome banding (CBA), several groups also confirmed CE with fluorescence in situ hybridization (FISH). At present, there are minimal prospective data on CE frequency determined using a combination of both methods. Therefore, the aim of our study was to prospectively assess CE frequency using a combination of FISH and CBA after stimulation with CpG oligonucleotides and interleukin-2. Between 2008 and 2012, we enrolled 140 patients with previously untreated CLL in a prospective trial evaluating CE using FISH and CBA after stimulation. Patients provided baseline and regular follow-up peripheral blood samples for testing. There was a median of 3 cytogenetic examinations (using both methods) per patient. CE was detected in 15.7% (22/140) of patients using FISH, in 28.6% (40/140) using CBA, and in 34.3% (48/140) of patients by combining both methods. Poor-prognosis CE (new deletion 17p, new deletion 11q or new complex karyotype) was detected in 15% (21/140) of patients and was significantly associated with previous CLL treatment (p=0.013). CBA provides more complex information about cytogenetic abnormalities in CLL patients than FISH and confirms that many patients can acquire new abnormalities during the course of their disease in a relatively short time period. PMID:24246692

Brejcha, Martin; Stoklasová, Martina; Brychtová, Yvona; Panovská, Anna; Št?panovská, Kristina; Va?ková, Gabriela; Plevová, Karla; Oltová, Alexandra; Horká, Kate?ina; Pospíšilová, Šárka; Mayer, Ji?í; Doubek, Michael

2014-02-01

318

Detection of numerical and structural alterations and fusion of chromosomes 16 and 1 in low-grade papillary breast carcinoma by fluorescence in situ hybridization.  

PubMed Central

Intracystic papillary breast tumors, including intraductal papilloma and low-grade intracystic papillary carcinoma, constitute a group for which differential diagnosis is frequently difficult. We examined the status of chromosomes 16 and 1 by multicolor fluorescence in situ hybridization (FISH) analyses and the DNA ploidy patterns by flow cytometry in 26 intracystic papillary tumors. Alterations of chromosomes 16 and 1 were detected by FISH in 93 and 85%, respectively, of 14 low-grade papillary carcinomas, and the latter alterations always concurred with the former. Two-color FISH using probes for the D1Z1 (1q12) and D16Z2 (16cen) loci or the D1Z1 and D16Z3 (16q11) loci showed that fusion of chromosomes 16 and 1, mostly with breakpoints distal to 16q11.2 and proximal to 1q12, occurred in 77% of the papillary carcinomas. DNA aneuploidy was detected in 6% of these carcinomas. No papilloma showed these chromosome alterations or DNA aneuploidy. Chromosome 16 and 1 fusions appeared to occur frequently in diploid breast carcinomas and to be involved in the acquisition of a malignant phenotype by duct epithelial cells. We suggest that two-color FISH methods for detecting 1;16 fusions might be applicable as supportive methods for the differential diagnosis of intracystic papillary breast tumors. Images Figure 1 Figure 2

Tsuda, H.; Takarabe, T.; Susumu, N.; Inazawa, J.; Okada, S.; Hirohashi, S.

1997-01-01

319

Assessment of Her-2/neu status using immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology smears: Experience from a tertiary care centre in India.  

PubMed

Breast carcinoma shows amplification/overexpression of Her-2/neu in ?20-30% of cases. The determination of Her-2/neu expression accurately is vital in clinical practice as it has significant predictive value and eligibility for anti Her-2/neu therapy. Amplification and overexpression of Her-2/neu gene is traditionally identified by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) on tissue sections; only a few studies have evaluated feasibility of these techniques on cytological smears. One hundred cases of breast cancer with fine-needle aspiration cytology (FNAC) samples and corresponding surgically resected specimen were selected. Immunocytochemistry (ICC) and FISH for Her-2/neu was done on FNA smears, whereas IHC was performed on corresponding tissue sections. Diagnostic accuracy of ICC was 99% when compared with IHC. Comparison of FISH results with IHC showed 100% concordance. Unlike many centers in West, FNAC is still routinely performed in developing countries like India where vast majority of breast cancer cases present as palpable lumps. The high rates of accuracy of ICC and FISH for Her-2/neu detection can make FNAC a relevant first line of investigation as a cost effective model with a rapid turn-around time, providing complete information necessary for initial management of breast cancer patients. Diagn. Cytopathol. 2014;42:726-731. © 2013 Wiley Periodicals, Inc. PMID:24376261

Durgapal, Prashant; Mathur, Sandeep R; Kalamuddin, Md; Datta Gupta, Siddhartha; Parshad, Rajinder; Julka, Pramod K; Panda, S K

2014-08-01

320

Radiation-induced chromosome aberrations in ataxia telangiectasia cells: high frequency of deletions and misrejoining detected by fluorescence in situ hybridization  

NASA Technical Reports Server (NTRS)

The mechanisms underlying the hyper-radiosensitivity of AT cells were investigated by analyzing chromosome aberrations in the G(2) and M phases of the cell cycle using a combination of chemically induced premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH) with chromosome painting probes. Confluent cultures of normal fibroblast cells (AG1522) and fibroblast cells derived from an individual with AT (GM02052) were exposed to gamma rays and allowed to repair at 37 degrees C for 24 h. At doses that resulted in 10% survival, GM02052 cells were approximately five times more sensitive to gamma rays than AG1522 cells. For a given dose, GM02052 cells contained a much higher frequency of deletions and misrejoining than AG1522 cells. For both cell types, a good correlation was found between the percentage of aberrant cells and cell survival. The average number of color junctions, which represent the frequency of chromosome misrejoining, was also found to correlate well with survival. However, in a similar surviving population of GM02052 and AG1522 cells, induced by 1 Gy and 6 Gy, respectively, AG1522 cells contained four times more color junctions and half as many deletions as GM02052 cells. These results indicate that both repair deficiency and misrepair may be involved in the hyper-radiosensitivity of AT cells.

Kawata, Tetsuya; Ito, Hisao; George, Kerry; Wu, Honglu; Uno, Takashi; Isobe, Kouichi; Cucinotta, Francis A.

2003-01-01

321

Genomic arrays in chronic lymphocytic leukemia routine clinical practice: are we ready to substitute conventional cytogenetics and fluorescence in situ hybridization techniques?  

PubMed

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Del(11q) and del(17p), routinely studied by conventional G-banding cytogenetics (CGC) and fluorescence in situ hybridization (FISH), have been related to progression and shorter overall survival. Recently, array-based karyotyping has gained acceptance as a high-resolution new tool for detecting genomic imbalances. The aim of the present study was to compare genomic arrays with CGC and FISH to ascertain whether the current techniques could be substituted in routine procedures. We analyzed 70 patients with CLL using the Cytogenetics Whole-Genome 2.7M Array and CytoScan HD Array (Affymetrix), CGC and FISH with the classical CLL panel. Whereas 31.4% and 68.6% of patients presented abnormalities when studied by CGC and FISH, respectively, these rates increased when arrays were also analyzed (78.6% and 80%). Although abnormality detection is higher when arrays are applied, one case with del(11q) and three with del(17p) were missed by genomic arrays due to their limited sensitivity. We consider that the complete substitution of CGC and FISH by genomic arrays in routine laboratories could negatively affect the management of some patients harboring 11q or 17p deletions. In conclusion, genomic arrays are valid to detect known and novel genomic imbalances in CLL, but should be maintained as a complementary tool to the current techniques. PMID:22994157

Puiggros, Anna; Puigdecanet, Eulŕlia; Salido, Marta; Ferrer, Ana; Abella, Eugčnia; Gimeno, Eva; Nonell, Lara; Herranz, María José; Galván, Ana Belén; Rodríguez-Rivera, María; Melero, Carme; Pairet, Silvia; Bellosillo, Beatriz; Serrano, Sergi; Florensa, Lourdes; Solé, Francesc; Espinet, Blanca

2013-05-01

322

Numerical aberrations of chromosomes 1 and 17 in tumor cell lines of the exocrine pancreas as determined by fluorescence in situ hybridization.  

PubMed

The relationship between the copy numbers of chromosomes 1 and 17 and characteristics related to aggressiveness, histological grade, and doubling time was studied in 10 cell lines from pancreatic carcinomas (7 from primary tumors, 2 from ascites, and 1 from a liver metastasis). Fluorescence in situ hybridization with, respectively, the (peri-)centromeric pUC 1.77 (chromosome 1), and D17Z1 (chromosome 17) probes was applied to interphase nuclei. The results showed that most of these cell lines were hyperploid for both chromosomes studied in accordance with their chromosome counts in metaphase; moreover, there existed a statistically significant positive correlation between the results for both probes. Two of these cell lines had a higher mean copy number for chromosome 17 than for chromosome 1 (PaCa 44 and PaTu 2); two of them had a higher mean copy number for chromosome 1 than for chromosome 17 (Panc 1 and PSN 1). Although only a weak correlation was observed between the number of signals for either chromosomes 1 or 17 and the doubling time, lines of grade 1 and 2 showed a lower average number of spots per nucleus than grade 3 cell lines for both chromosomes studied. PMID:9109941

Verdoodt, B; Charlette, I; Maillet, B; Kirsch-Volders, M

1997-04-01

323

High-Resolution Single-Copy Gene Fluorescence in Situ Hybridization and Its Use in the Construction of a Cytogenetic Map of Maize Chromosome 9[W  

PubMed Central

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by ?100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize.

Wang, Chung-Ju Rachel; Harper, Lisa; Cande, W. Zacheus

2006-01-01

324

High-resolution single-copy gene fluorescence in situ hybridization and its use in the construction of a cytogenetic map of maize chromosome 9.  

PubMed

High-resolution cytogenetic maps provide important biological information on genome organization and function, as they correlate genetic distance with cytological structures, and are an invaluable complement to physical sequence data. The most direct way to generate a cytogenetic map is to localize genetically mapped genes onto chromosomes by fluorescence in situ hybridization (FISH). Detection of single-copy genes on plant chromosomes has been difficult. In this study, we developed a squash FISH procedure allowing successful detection of single-copy genes on maize (Zea mays) pachytene chromosomes. Using this method, the shortest probe that can be detected is 3.1 kb, and two sequences separated by approximately 100 kb can be resolved. To show the robust nature of this protocol, we localized nine genetically mapped single-copy genes on chromosome 9 in one FISH experiment. Integration of existing information from genetic maps and the BAC contig-based physical map with the cytological structure of chromosome 9 provides a comprehensive cross-referenced cytogenetic map and shows the dramatic reduction of recombination in the pericentromeric heterochromatic region. To establish a feasible mapping system for maize, we also developed a probe cocktail for unambiguous identification of the 10 maize pachytene chromosomes. These results provide a starting point toward constructing a high-resolution integrated cytogenetic map of maize. PMID:16461583

Wang, Chung-Ju Rachel; Harper, Lisa; Cande, W Zacheus

2006-03-01

325

Vertical distribution of Archaea and Bacteria in a meromictic lake as determined by fluorescent in situ hybridization.  

PubMed

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A "red-water" layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem. PMID:22006072

Lentini, Valeria; Gugliandolo, Concetta; Maugeri, Teresa L

2012-01-01

326

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.  

PubMed

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 = 7), pure diploid (n = 7), or pure haploid (n = 2). The remaining 16 embryos showed mosaic patterns; 1 was triploid-diploid mosaics, 9 were diploid-haploid, and 4 were haploid only. Autosomal aneuploidy occurred in 2 embryos showing a triploid complement. The sex chromosomal constituent XXX:XXY:XYY was 3:4:2 in embryos showing a pure triploid complement (including 2 embryos with aneuploidy). This ratio was not significantly different from the expected 1:2:1 (p = 0.96). Pure triploid was found in 41.7% of 2-cell embryos, but no triploid complement was found in 3-cell embryos. The present study also supports the diandric origin of tripronuclear zygotes in the conventional IVF, and, to our knowledge, is the first study to use simultaneous six-color FISH for chromosomes 4, 13, 18, 21, X, and Y in human embryos. However, no additive information was obtained about chromosome 4. PMID:15627776

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

2005-01-01

327

Detection of Her2/neu, c-MYC and ZNF217 gene amplification during breast cancer progression using fluorescence in situ hybridization.  

PubMed

The deletion of tumor suppressor genes and amplification of activating oncogenes appear to be critical events in tumorigenesis. We carried out fluorescence in situ hybridization (FISH) analysis of the breast cancer cell line MCF7 and clinically obtained cancer tissue sections on the basis of which we suggest a breast cancer development model. We selected 28 genes for FISH probes from breast cancer patients. Of the 28 genes studied, 14 were shown to be consistently amplified in the breast cancer cell line MCF7. We selected three genes from clinical tumor samples on the basis of results from MCF7 analysis. The amplification of Her2/neu or ZNF217 is closely associated with the stages of breast cancer. The frequency of amplification of Her2/neu increased notably in patients at stages later than IIB based on TNM staging, whereas the amplification of ZNF217 correlated with T2N1M0 at stage IIB and later stages. c-MYC amplification was not related to the stage. Her2/neu, ZNF217 and c-MYC were found to have a significantly increased copy number in breast cancer cells. In breast cancer progression, c-MYC amplification is an early event, while ZNF217 and Her2/neu amplification may play a role in the later stage of tumor development. PMID:15756435

Shimada, Masayuki; Imura, Johji; Kozaki, Takazumi; Fujimori, Takahiro; Asakawa, Shuichi; Shimizu, Nobuyoshi; Kawaguchi, Ryuji

2005-04-01

328

Chromosome anomalies detected by interphase fluorescence in situ hybridization: correlation with significant biological features of B-cell chronic lymphocytic leukaemia.  

PubMed

Fluorescence in situ hybridization (FISH) was used to detect 6q-, 11q-, +12, 13q-, 17p- and translocations involving 14q32 in interphase nuclei from blood and/or bone marrow from 113 patients with B-cell chronic lymphocytic leukaemia (B-CLL). A total of 87 patients (77%) had a FISH anomaly: 13q- x 1 was most frequent (64%) followed by 13q- x 2 (28%), +12 (25%), 11q- (15%), 17p- (8%) and 6q- (0%). FISH results for blood and bone marrow cells in 38 patients were similar. Purified CD5+/CD19+ cells from blood were studied in eight patients and results indicate that in some patients not all B cells have FISH anomalies. We used a defined set of hierarchical FISH risk categories to compare FISH results by stable versus progressive disease, age, sex, Rai stage, CD38+ expression and IgVH mutational status. Significant differences in FISH risk distributions were associated with Rai stage, disease status and CD38+, but not by age, sex or IgVH mutational status. To look for baseline factors associated with high-risk disease, multivariate analysis of age, sex, Rai stage, CD38+ and disease status versus FISH risk category was performed. Importantly, only CD38+ was significantly associated with high-risk FISH categories (+12, 11q- and 17p-) after adjustment for the effects of other variables. PMID:12694251

Dewald, Gordon W; Brockman, Stephanie R; Paternoster, Sarah F; Bone, Nancy D; O'Fallon, Judith R; Allmer, Cristine; James, Charles D; Jelinek, Diane F; Tschumper, Renee C; Hanson, Curtis A; Pruthi, Rajiv K; Witzig, Thomas E; Call, Timothy G; Kay, Neil E

2003-04-01

329

The detection of hTERC amplification using fluorescence in situ hybridization in the diagnosis and prognosis of cervical intraepithelial neoplasia: a case control study  

PubMed Central

Background Currently the routine non-invasive screening methods for cervical intraepithelial neoplasia (CIN) and cervical cancer are Thinprep cytology test (TCT) and human papillomavirus testing. However, both methods are limited by the high false positive and false negative rates and lack of association with patients’ prognosis, especially for the early detection of pro-malignant CIN. The aim of the study was to investigate the role of genomic amplification of human telomerase gene (hTERC) in the diagnosis and prognosis of CIN. Methods The study group consisted of specimens of exfoliated cervical cells from 151 patients, including 27 with CIN I, 54 with CIN II/III, 17 with carcinoma in situ, and 28 with invasive squamous carcinoma, as well as 25 patients who were at 2-year follow-up after either Loop Electrosurgical Excision treatment (n?=?11) or radical surgery (n?=?14). hTERC amplification was detected by dual-color interphase fluorescence in situ hybridization (FISH), and the results were compared with TCT and histologic examination. The final diagnosis was determined by the pathological examination. The control group consisted of specimens of exfoliated cervical cells from 40 normal women. Results The percentage of cervical exfoliated cells with positive hTERC amplification and incidence rates of hTERC amplification were 9.2%?±?4.6% and 44.4% (12/27) respectively in patients with CIN I; 16.0%?±?14.4% and 85.1% (46/54) in patients with CIN II/III; 19.7%?±?13.3% and 88.3% (15 /17) in patients with carcinoma in situ; 47.0%?±?25.2% and 100% (28/28)in patients with invasive squamous carcinoma. There was statistically significant difference between the control and study group (P <0.01), and between the patients with various diseases within the study group (P <0.05). Conclusion The detection of genomic amplification of hTERC using FISH is a non-invasive and effective approach for CIN.

2012-01-01

330

Comparison of high resolution chromosome banding and fluorescence in situ hybridization (FISH) for the laboratory evaluation of Prader-Willi syndrome and Angelman syndrome  

SciTech Connect

The development of probes containing segments of DNA from chromosome region 15q11-q13 provides the opportunity to confirm the diagnosis of Prader-Willi syndrome (PWS) and Angelman syndrome (AS) by fluorescence in situ hybridization (FISH). We have evaluated FISH studies and high resolution chromosome banding studies in 14 patients referred to confirm or rule out AS. In four patients (three from the PWS category and 1 from the AS group) chromosome analysis suggested that a deletion was present but FISH failed to confirm the finding. In one AS group patient, FISH identified a deletion not detectable by high resolution banding. Review of the clinical findings in the discrepant cases suggested that FISH results were correct and high resolution findings were erroneous. Studies with a chromosome 15 alpha satellite probe (D15Z) on both normal and abnormal individuals suggested that incorrect interpretation of chromosome banding may occasionally be attributable to alpha satellite polymorphism but other variation of 15q11-q13 chromosome bands also contributes to misinterpretation. We conclude that patients who have been reported to have a cytogenetic deletion of 15q11-q13 and who have clinical findings inconsistent with PWS and AS should be re-evaluated by molecular genetic techniques. 31 refs., 3 figs., 2 tabs.

Delach, J.A.; Rosengren, S.S.; Kaplan, L.; Greenstein, R.M.; Cassidy, S.B.; Benn, P.A.

1994-08-01

331

The human ovarian teratocarcinoma cell line PA-1 demonstrates a single translocation: analysis with fluorescence in situ hybridization, spectral karyotyping, and bacterial artificial chromosome microarray.  

PubMed

Cell lines derived from tumors contain numerous chromosomal aberrations and are the focus of study in tumor evolution. The ovarian teratocarcinoma cell line PA-1 demonstrates a single chromosomal aberration: a reciprocal t(15;20)(p11.2;q11.2). A complete molecular genetic analysis was undertaken to characterize this cell line. The PA-1 cell line was studied with fluorescence in situ hybridization (FISH), spectral karyotyping (SKY), bacterial artificial chromosome (BAC) microarray, and Western blotting. Amplification of 20q is frequently implicated in both breast and ovarian cancer; this region contains a number of oncogenes including MDM2, ZNF217, and the ovarian tumor marker WFDC2 (alias HE4). FISH revealed gene amplification of AIB1 (now known as NCOA3) but not STK15 (now known as AURKA). Immunoblot analysis demonstrated 3.6-fold overexpression of the AIB1 protein product, but no elevation of the STK15. BAC cancer gene microarray analysis showed gene amplification of > or =1.20 for five oncogenes. The presence of a consistent single change in PA-1, the t(15;20)(p11.2;q11.2), suggests that the aberration is significant with respect to the transformation status of the cell line. This translocation appears to cause overexpression of AIB1 (and perhaps other proteins), which may provide an immortalizing effect on this cell line. PMID:16080959

Sarraf, Shireen; Tejada, Raphael; Abawi, Massih; Oberst, Michael; Dennis, Tom; Simon, Kelly Claire; Blancato, Jan

2005-08-01

332

Fluorescence in situ hybridization with specific oligonucleotide rRNA probes distinguishes the sibling species Stylonychia lemnae and Stylonychia mytilus (Ciliophora, Spirotrichea).  

PubMed

Based on morphological and morphogenetic characters alone, the sibling species Stylonychia lemnae and Stylonychia mytilus, members of the Stylonychia mytilus complex, can hardly be distinguished. However, biochemical investigations of the isoenzyme pattern of different enzymes showed a distinct differentiation between these two species. In the last few years, fluorescence in situ hybridization (FISH) techniques have become a suitable and reliable tool for identification and differentiation of closely related species of protozoa, such as ciliates. To distinguish the sibling species, a set of specific oligonucleotide probes were developed. In the present study, the SSU rDNA of 7 clones of Stylonychia lemnae and 13 clones of Stylonychia mytilus, isolated from different geographic regions, were sequenced. Comparing all SSU rDNA sequences of both species, only one single difference within the whole gene was detected. Based on this difference, a set of two oligonucleotide probes, targeting the SSU rRNA of each species (Stylonychia mytilus and Stylonychia lemnae) was designed. These probes were successfully tested by applying the FISH techniques on preserved cells of different clones of both species. PMID:16427805

Schmidt, Stephanie L; Bernhard, Detlef; Schlegel, Martin; Fried, Johannes

2006-02-01

333

Assignment of the human prostaglandin-endoperoxide synthase 2 (PTGS2) gene to 1q25 by fluorescence in situ hybridization  

SciTech Connect

A major mechanism for the regulation of prostaglandin synthesis occurs at the level of cyclooxygenase, also known as prostaglandin-endoperoxide synthase (PTGS). Two isoforms of PTGS have been identified: PTGS1, encoded by a 2.8-kb mRNA and a mitogen-inducible form, PTGS2, encoded by a 4.5-kb mRNA. We report here the assignment of the human PTGS2 gene to chromosome 1q25 by fluorescence in situ hybridization (FISH). We note with interest the physical proximity of the PTGS2 gene to that of cytosolic phospholipase A2 (cPLA2), which we have previously mapped to chromosome 1q24-q25. In contrast, the PTGS1 gene has been mapped to chromosome 9. Since cPLA2 and PTGS2 are key enzymes in the synthesis of prostaglandins and thromboxane, the possible implication of this proximity could mean that polymorphic markers already determined for the cPLA2 gene may also prove to be useful as markers for the PTGS2 gene as well. 10 refs., 1 fig.

Tay, A.; Squire, J.A.; Goldberg, H.; Skorecki, K. [Univ. of Toronto (Canada)] [Univ. of Toronto (Canada)

1994-10-01

334

Two congenital cases of pigmented epithelioid melanocytoma studied by fluorescent in situ hybridization for melanocytic tumors: case reports and review of these recent topics.  

PubMed

The term 'pigmented epithelioid melanocytoma' (PEM) has recently been proposed as a nosological framework grouping lesions formerly known as animal-type melanomas, sporadic epithelioid blue nevi and Carney complex-associated epithelioid blue nevi. Congenital PEMs have been reported extremely rarely and their prognosis is poorly known. Four-color fluorescent in situ hybridization (FISH) for melanocytic lesions is a recent method developed to assess the malignant potential of ambiguous melanocytic lesions. Here we describe 2 cases of congenital epithelioid and strongly pigmented melanocytic lesions consistent with PEM. No BRAF gene mutation was found in the 2 cases. FISH for melanocytic lesions was also performed. The first case proved entirely negative, whereas the second one showed a positive zone with an extra copy of chromosome 6. The prognosis and management of PEM are discussed, with a review of the available data on the history, demographics, molecular alterations and histopathological aspects of this entity. PEM seems to represent a unique low-grade melanocytic tumor with a limited potential of metastasis to lymph nodes, but a favorable long-term clinical course. The published data about FISH for melanocytic tumors, and especially PEM, are reviewed. Four-color FISH may be a useful tool to assess more accurately the prognosis of these tumors. PMID:20558976

Battistella, M; Prochazkova-Carlotti, M; Berrebi, D; Bennaceur, S; Edan, C; Riffaud, L; Rütten, A; Fraitag, S

2010-01-01

335

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

PubMed Central

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.

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

2014-01-01

336

Image analysis of HER2 immunohistochemical staining. Reproducibility and concordance with fluorescence in situ hybridization of a laboratory-validated scoring technique.  

PubMed

Image analysis of the HER2 immunohistochemical (IHC) stain can help determine which breast cancer patients may benefit from HER2-targeted therapy. We studied the concordance of HER2 IHC and fluorescence in situ hybridization (FISH) as well as reproducibility of surgical pathologist (SP) and cytotechnologist (CT) interpretations using manual and image analysis methodologies on 154 IHC cases. Concordances with FISH were good for IHC negative (0, 1+) cases (range, 97%-100%) and positive (3+) cases (range, 87%-100%). Image analysis had fewer equivocal (2+) results (10.4%) than CT (14.9%) and SP (16.2%) manual methods, with higher concordances to FISH (31%, 26%, and 20% for image analysis, CT manual, and SP manual, respectively). CT manual (? = 0.747) and image analysis (? = 0.779) methods had better interobserver reproducibility than SP manual (? = 0.697). CT image analysis had better intraobserver reproducibility (? = 0.882) than CT (? = 0.828) and SP (? = 0.766) manual methods. HER2 IHC analysis performed by image analysis can produce accurate results with improved reproducibility. PMID:22261453

Minot, Douglas M; Voss, Jesse; Rademacher, Susan; Lwin, Toe; Orsulak, Jessica; Caron, Bolette; Ketterling, Rhett; Nassar, Aziza; Chen, Beiyun; Clayton, Amy

2012-02-01

337

Specific Detection and Prevalence of Helicobacter heilmannii-Like Organisms in the Human Gastric Mucosa by Fluorescent In Situ Hybridization and Partial 16S Ribosomal DNA Sequencing  

PubMed Central

Gastric infection with Helicobacter heilmannii (previously known as Gastrospirillum hominis) is invariably linked with the presence of chronic gastritis and the risk of developing low-grade mucosa-associated lymphoid tissue lymphoma in humans. In contrast to Helicobacter pylori, various H. heilmannii species colonize the stomachs of domestic animals, which might be a reservoir for transmission to humans (zoonosis). To identify the number and prevalence of different H. heilmanni types in humans, we analyzed 89 gastric biopsy samples histologically identified as H. heilmannii positive by fluorescence in situ hybridization. Of these gastric specimens, 84 (94.4%) contained a single H. heilmannii type. In five samples, however, two different H. heilmannii types were detected. The most prevalent species in monoinfected samples is H. heilmannii type 1, found in 78.5% (66 of 84) of the specimens, followed by a novel H. heilmannii-like organism (HHLO), HHLO type 4, identified in 9.6% (8 of 84) of tissue sections. H. heilmannii type 2 and a further HHLO type not described before, type 3, were found in 8.3% (7 of 84) and 1.2% (1 of 84) of the monoinfected samples, respectively. Additionally, HHLO type 5 with a 16S ribosomal DNA sequence identical to that of Helicobacter salomonis was found with a prevalence of 2.4% (2 of 89). Thirteen of these biopsy samples were also investigated by a PCR approach developed for this study that allows a Helicobacter-specific amplification of a variable portion of the 16S rRNA gene and subsequent sequencing. In total, five different types of HHLOs could be identified within these samples. We conclude that humans can be infected by at least five different HHLO types, which presumably have their origin in animal species like dogs, cats, and pigs.

Trebesius, K.; Adler, K.; Vieth, M.; Stolte, M.; Haas, R.

2001-01-01

338

Specific detection and prevalence of Helicobacter heilmannii-like organisms in the human gastric mucosa by fluorescent in situ hybridization and partial 16S ribosomal DNA sequencing.  

PubMed

Gastric infection with Helicobacter heilmannii (previously known as Gastrospirillum hominis) is invariably linked with the presence of chronic gastritis and the risk of developing low-grade mucosa-associated lymphoid tissue lymphoma in humans. In contrast to Helicobacter pylori, various H. heilmannii species colonize the stomachs of domestic animals, which might be a reservoir for transmission to humans (zoonosis). To identify the number and prevalence of different H. heilmanni types in humans, we analyzed 89 gastric biopsy samples histologically identified as H. heilmannii positive by fluorescence in situ hybridization. Of these gastric specimens, 84 (94.4%) contained a single H. heilmannii type. In five samples, however, two different H. heilmannii types were detected. The most prevalent species in monoinfected samples is H. heilmannii type 1, found in 78.5% (66 of 84) of the specimens, followed by a novel H. heilmannii-like organism (HHLO), HHLO type 4, identified in 9.6% (8 of 84) of tissue sections. H. heilmannii type 2 and a further HHLO type not described before, type 3, were found in 8.3% (7 of 84) and 1.2% (1 of 84) of the monoinfected samples, respectively. Additionally, HHLO type 5 with a 16S ribosomal DNA sequence identical to that of Helicobacter salomonis was found with a prevalence of 2.4% (2 of 89). Thirteen of these biopsy samples were also investigated by a PCR approach developed for this study that allows a Helicobacter-specific amplification of a variable portion of the 16S rRNA gene and subsequent sequencing. In total, five different types of HHLOs could be identified within these samples. We conclude that humans can be infected by at least five different HHLO types, which presumably have their origin in animal species like dogs, cats, and pigs. PMID:11283079

Trebesius, K; Adler, K; Vieth, M; Stolte, M; Haas, R

2001-04-01

339

Visualization of 'Candidatus Liberibacter asiaticus' cells in the vascular bundle of citrus seed coats with fluorescence in situ hybridization and transmission electron microscopy.  

PubMed

'Candidatus Liberibacter asiaticus' is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for 'Ca. L. asiaticus' were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the 'Ca. L. asiaticus' 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to 'Ca. L. asiaticus'. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no 'Ca. L. asiaticus' sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as 'Ca. L. asiaticus', providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were 'Ca. L. asiaticus'. PMID:23676087

Hilf, Mark E; Sims, Kenneth R; Folimonova, Svetlana Y; Achor, Diann S

2013-06-01

340

Correlation of cytomorphology, immunophenotyping, and interphase fluorescence in situ hybridization in 381 patients with monoclonal gammopathy of undetermined significance and 301 patients with plasma cell myeloma.  

PubMed

To further clarify the transformation from monoclonal gammopathy of undetermined significance (MGUS) to plasma cell myeloma (PCM), we compared interphase fluorescence in situ hybridization (FISH) patterns in 381 MGUS and 301 PCM patients. According to the World Health Organization and the International Myeloma Working Group, a threshold of 10% of bone marrow plasma cells separated MGUS from PCM. After magnetic activated cell sorting for CD138(+) cells, FISH succeeded in 272 of 301 (90.4%) PCM, but in only 302 of 381 (79.3%) MGUS cases (P < 0.001). Cytogenetic alterations were more frequent in PCM (237 of 272; 87.1%) than MGUS (169 of 302; 56.0%; P = 0.0002). PCM showed a median of two cytogenetic alterations (range, 0-9) and MGUS one (range, 0-6). Considering only cases with a yield of plasma cells allowing five or more FISH probes, del(13)(q14) was found in 99 of 251 (39.3%) PCM but in only 59 of 267 (22.1%) MGUS (P = 0.0001), del(17p) in 15 PCM (6.0%) and in 6 MGUS (2.2%) patients (P = 0.029). A t(4;14)/IGH-FGFR3 was detected in 28 PCM (11.1%) and 5 MGUS (1.9%; P < 0.001). The t(11;14)/IGH-CCND1 and the t(14;16)/IGH-MAF showed no significant differences. Cytomorphology detected higher numbers of plasma cells than multiparameter flow cytometry (median ratio 4.25). This study underlines the genetic heterogeneity of MGUS similar to PCM. Genetic analysis might contribute to more diversified monitoring strategies for MGUS patients. PMID:21156229

Bacher, Ulrike; Haferlach, Torsten; Kern, Wolfgang; Alpermann, Tamara; Schnittger, Susanne; Haferlach, Claudia

2010-12-01

341

Nucleic acid in-situ hybridization detection of infectious agents  

NASA Astrophysics Data System (ADS)

Limitations of traditional culture methods and newer polymerase chain reaction (PCR)-based methods for detection and speciation of infectious agents demonstrate the need for more rapid and better diagnostics. Nucleic acid hybridization is a detection technology that has gained wide acceptance in cancer and prenatal cytogenetics. Using a modification of the nucleic acid hybridization technique known as fluorescence in-situ hybridization, infectious agents can be detected in a variety of specimens with high sensitivity and specificity. The specimens derive from all types of human and animal sources including body fluids, tissue aspirates and biopsy material. Nucleic acid hybridization can be performed in less than one hour. The result can be interpreted either using traditional fluorescence microscopy or automated platforms such as micro arrays. This paper demonstrates proof of concept for nucleic acid hybridization detection of different infectious agents. Interpretation within a cytologic and histologic context is possible with fluorescence microscopic analysis, thereby providing confirmatory evidence of hybridization. With careful probe selection, nucleic acid hybridization promises to be a highly sensitive and specific practical diagnostic alternative to culture, traditional staining methods, immunohistochemistry and complicated nucleic acid amplification tests.

Thompson, Curtis T.

2000-04-01

342

Evidence of Multiple Treponema Phylotypes Involved in Bovine Digital Dermatitis as Shown by 16S rRNA Gene Analysis and Fluorescence In Situ Hybridization?  

PubMed Central

The etiopathogenesis of the skin disease digital dermatitis (DD), an important cause of lameness in cattle, remains uncertain. Microscopically, the disease appears to be polymicrobial, with spirochetes as the predominant bacteria. The objective of this study was to identify the main part of the bacteria involved in DD lesions of cattle by using culture-independent molecular methods. Ten different phylotypes of Treponema were identified either by 16S rRNA gene sequencing of bacteria from DD lesions or by fluorescence in situ hybridization (FISH) analysis using phylotype-specific 16S rRNA-directed oligonucleotide probes. Two phylotypes, phylotype 1 (PT1) and PT2, were not closely related to any characterized treponemal species. PT7 was 99.3% identical to Treponema denticola, while PT9 resembled T. vincentii by 96%. The remaining phylotypes, PT3, PT4, PT5, PT6, and PT8, and Treponema brennaborense had previously been isolated from DD lesions. Forty DD biopsy specimens were examined for Treponema by FISH. With one exception, all of the biopsy specimens revealed epidermotropic, intermingled infection with three or more different phylotypes (mean, 4.7). The most prevalent species were PT1 (95%), PT6 (93%), and PT3 (85%). While colonization by PT3 was confined to the surface of the epidermis, both PT1 and PT6 invaded deep into the stratum spinosum and were seen in ulcerated dermal papillae. In two cases, all 10 phylotypes were demonstrated. Furthermore, FISH with a Treponema group-specific probe showed that Treponema accounted for more than 90% of the total bacterial population in the biopsy specimens. These data strongly suggest that a group of apparently symbiotic Treponema species are involved as primary bacterial pathogens in DD.

Klitgaard, Kirstine; Boye, Mette; Capion, Nynne; Jensen, Tim K.

2008-01-01

343

Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Salmonella spp. in a broad spectrum of samples.  

PubMed

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 x 10(9) +/- 5 x 10(8) CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 x 10(7) +/- 5 x 10(6) CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4',6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity. PMID:20453122

Almeida, C; Azevedo, N F; Fernandes, R M; Keevil, C W; Vieira, M J

2010-07-01

344

Fluorescence In Situ Hybridization Method Using a Peptide Nucleic Acid Probe for Identification of Salmonella spp. in a Broad Spectrum of Samples ?  

PubMed Central

A fluorescence in situ hybridization (FISH) method for the rapid detection of Salmonella spp. using a novel peptide nucleic acid (PNA) probe was developed. The probe theoretical specificity and sensitivity were both 100%. The PNA-FISH method was optimized, and laboratory testing on representative strains from the Salmonella genus subspecies and several related bacterial species confirmed the predicted theoretical values of specificity and sensitivity. The PNA-FISH method has been successfully adapted to detect cells in suspension and is hence able to be employed for the detection of this bacterium in blood, feces, water, and powdered infant formula (PIF). The blood and PIF samples were artificially contaminated with decreasing pathogen concentrations. After the use of an enrichment step, the PNA-FISH method was able to detect 1 CFU per 10 ml of blood (5 × 109 ± 5 × 108 CFU/ml after an overnight enrichment step) and also 1 CFU per 10 g of PIF (2 × 107 ± 5 × 106 CFU/ml after an 8-h enrichment step). The feces and water samples were also enriched according to the corresponding International Organization for Standardization methods, and results showed that the PNA-FISH method was able to detect Salmonella immediately after the first enrichment step was conducted. Moreover, the probe was able to discriminate the bacterium in a mixed microbial population in feces and water by counter-staining with 4?,6-diamidino-2-phenylindole (DAPI). This new method is applicable to a broad spectrum of samples and takes less than 20 h to obtain a diagnosis, except for PIF samples, where the analysis takes less than 12 h. This procedure may be used for food processing and municipal water control and also in clinical settings, representing an improved alternative to culture-based techniques and to the existing Salmonella PNA probe, Sal23S10, which presents a lower specificity.

Almeida, C.; Azevedo, N. F.; Fernandes, R. M.; Keevil, C. W.; Vieira, M. J.

2010-01-01

345

Evidence for Cytogenetic and Fluorescence In Situ Hybridization Risk Stratification of Newly Diagnosed Multiple Myeloma in the Era of Novel Therapies  

PubMed Central

Overall survival (OS) has improved with increasing use of novel agents in multiple myeloma (MM). However, the disease course remains highly variable, and the heterogeneity largely reflects different genetic abnormalities. We studied the impact of the Mayo risk-stratification model of MM on patient outcome in the era of novel therapies, evaluating each individual component of the model—fluorescence in situ hybridization (FISH), conventional cytogenetics (CG), and the plasma cell labeling index—that segregates patients into high- and standard-risk categories. This report consists of 290 patients with newly diagnosed MM, predominantly treated with novel agents, who were risk-stratified at diagnosis and were followed up for OS. Of these patients, 81% had received primarily thalidomide (n=50), lenalidomide (n=199), or bortezomib (n=79) as frontline or salvage therapies. Our retrospective analysis validates the currently proposed Mayo risk-stratification model (median OS, 37 months vs not reached for high- and standard-risk patients, respectively; P=.003). Although the FISH or CG test identifies a high-risk cohort with hazard ratios of 2.1 (P=.006) and 2.5 (P=.006), respectively, the plasma cell labeling index cutoff of 3% fails to independently prognosticate patient risk (hazard ratio, 1.4; P=.41). In those stratified as standard-risk by one of the 2 tests (FISH or CG), the other test appears to be of additional prognostic significance. This study validates the high-risk features defined by FISH and CG in the Mayo risk-stratification model for patients with MM predominantly treated with novel therapies based on immunomodulatory agents.

Kapoor, Prashant; Fonseca, Rafael; Rajkumar, S. Vincent; Sinha, Shirshendu; Gertz, Morie A.; Stewart, A. Keith; Bergsagel, P. Leif; Lacy, Martha Q.; Dingli, David D.; Ketterling, Rhett P.; Buadi, Francis; Kyle, Robert A.; Witzig, Thomas E.; Greipp, Philip R.; Dispenzieri, Angela; Kumar, Shaji

2010-01-01

346

Low or absent unconjugated estriol in pregnancy: an indicator for steroid sulfatase deficiency detectable by fluorescence in situ hybridization and biochemical analysis.  

PubMed

It has been previously reported that a low or absent maternal serum unconjugated estriol (uE3) level is associated with placental steroid sulfatase (STS) deficiency. Here we report a correlation between patients who present with a very low or absent maternal serum uE3 and a deletion of the STS gene as assessed by fluorescence in situ hybridization (FISH). We studied nine prenatal cases that presented to the clinical laboratory with an abnormal triple screen, specifically low or absent maternal serum uE3 and a 46,XY karyotype. FISH analysis showed complete deletion of a probe containing the STS gene in six cases and one case had a partial deletion (reduced but not absent signal). The remaining two cases were not deleted for the STS probe. All mothers tested whose fetus showed a deletion were shown to be STS deletion carriers using FISH. Biochemical analysis was performed on 7/9 prenatal specimens. All fetuses deleted for the STS probe were also found to be deficient for STS by biochemical analysis of cultured amniotic fluid (5/5). Of the two fetuses not deleted for the STS probe, one was deficient for STS activity, while the other had a normal result. The abnormal result of enzyme deficiency by biochemical analysis in a non-deletion case likely represents a mutation in the STS gene, not detectable by this FISH assay. Postnatal FISH confirmation of the STS deletion was performed in 1/7 cases. Clinical follow-up was available for 4/9 cases following birth. PMID:12424769

Kashork, Catherine D; Sutton, V Reid; Fonda Allen, Jill S; Schmidt, Deborah E; Likhite, Marisa L; Potocki, Lorraine; O'Brien, William E; Shaffer, Lisa G

2002-11-01

347

Large-scale polymorphism near the ends of several human chromosomes analyzed by using fluorescence in situ hybridization (FISH)  

SciTech Connect

We have discovered a large DNA segment that is polymorphically present at the ends of several human chromosomes. The segment, f7501, was originally derived form a human chromosome 19-specific cosmid library. FISH was used to determine the cosmid`s chromosomal distribution on 44 unrelated humans and several closely related primates. The human subjects represent a diversity of reproductively isolated ethnic populations. FISH analysis revealed that sequences highly homologous to the cosmid`s insert are present on both homologs at 3q, 15q,. and 19p in almost all individuals (88, 85, and 87 of 88 homologs, respectively). Other chromosomes sites were labeled much more rarely in the sampled individuals. For example, 56 of the 88 analyzed chromosomes 11 were labeled (18+/+, 6-/-, and 20+/- individuals). In contrast, 2q was labeled on only 1/88 sampled chromosomes. The termini of 2q, 5q, 6p, 6q, 7p, 8p, 9p, 9q, 11p, 12q, 16p, 19q, and 20q and an interstitial site at 2q13-14 were labeled in at least one individual of the set. EcoR1-fragments derived from the cosmid showed the same hybridization pattern as the entire cosmid, indicating that at least 40 kbp is shared by these chromosome ends. Ethnic differences in the allele frequency of these polymorphic variants was observed. For example, signals were observed on 8/10 and 7/10 of the chromosomes 7p and 16q, respectively, derived form Biakan Pygmies, but these sites were infrequently labeled in non-Pygmy human populations (2/68, respectively). This region has undergone significant changes in chromosome location during human evolution. Strong signal was seen on chimpanzee and gorilla chromosome 3, which is homologous to human chromosome 4, a chromosome unlabeled in any of the humans we have analyzed.

Trask, B.J.; Friedman, C. [Univ. of Washington, Seattle, WA (United States); Giorgi, D. [CNRS, Montpelier (France)] [and others

1994-09-01

348

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

SciTech Connect

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.

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

349

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  

PubMed Central

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 DNA simultaneously relative to total bacterial DNA. The assay has a high discriminatory power and resolves concentration changes as low as 1.3-fold. The methodology was compared with flow cytometric fluorescence in situ hybridization (FLOW-FISH) and 5-bromo-4-chloro-3-indolyl-?-d-galactopyranoside (X-Gal)-calcium citrate agar-based plate counting. For enumeration by FLOW-FISH, three new probes having the same specificity as the qPCR assay were designed and established. A combination with flow cytometry greatly reduced the time consumed compared to manual enumeration. Both qPCR and FLOW-FISH yielded similar community compositions for 10 complex starter cultures, with all detected subpopulations being highly significantly correlated (P < 0.001). Correlations between X-Gal-calcium citrate agar-based CFU and qPCR-derived counts were highly significant (P < 0.01 and P < 0.001, respectively) for the number of acidifiers versus L. lactis subsp. cremoris and for Leuconostoc spp. as quantified by the two techniques, respectively. This confirmed that most acidifiers in the studied PROBAT cultures are members of L. lactis subsp. cremoris. Quantitative real-time PCR and FLOW-FISH were found to be effective and accurate tools for the bacterial community analysis of complex starter cultures.

Friedrich, Udo; Lenke, Jan

2006-01-01

350

Mixed mullerian tumor of endometrium: A case study with sixteen DNA probes using fluorescent in situ hybridization  

Microsoft Academic Search

Summary Carcinosarcomas (previously identified as Mixed Mullerian Tumors, or MMT) of the uterus are uncommon and complex malignancies. Cytogenetic studies of these tumors are few and molecular investigations of their oncogenes are rare. We present a case including a study of this tumor with a panel of eleven fluorescent DNA probes of PML\\/RAR!, RB-13q, c-myc, 20q13.2 (ZNF217), inv 16, Cyclin-D,

Shamim A. Faruqi; Holly Prescott; Christopher Harsch; Harvey Spector; Joel S. Noumoff

2008-01-01

351

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

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.

2013-01-01

352

Characterization of Childhood Precursor T-Lymphoblastic Lymphoma by Immunophenotyping and Fluorescent In-Situ Hybridization: A Report from the Children's Oncology Group  

PubMed Central

Background T-lymphoblastic lymphoma (T-LBL) accounts for 25–30% of childhood non-Hodgkin’s lymphoma and is closely related to T-lymphoblastic leukemia (T-ALL). Recently, we demonstrated distinct differences in gene expression between childhood T-LBL and T-ALL, but molecular pathogenesis and relevant protein expression patterns in T-LBL remain poorly understood. Procedure Children with T-LBL with disseminated disease were registered and treated on COG protocol 5971. Paraffin-embedded tumor tissue was obtained at diagnosis for immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) studies. We determined the pattern and intensity of staining for c-Myc, Skp2, Mib-1, p53, TCL-1, bcl-2, and bcl-6 proteins by IHC and c-Myc, p53, bcl-2, bcl-6, and TCR ?/? molecular alterations by FISH in 22 pediatric T-LBL cases. Results The majority of T-LBL samples expressed Mib-1 (59%) and c-Myc (77%) proteins in greater than 50% of the cells, but Skp2 (14%), p53 (14%), and bcl-2 (23%) expression was less common. FISH studies demonstrated 18% gains and 10% losses in c-Myc, 16% gains in p53, 12% gains and 6% losses in bcl-2, and 6% gains and 19% losses in bcl-6 with little direct correlation between the IHC and FISH studies. Conclusions Childhood T-LBL is a highly proliferative tumor associated with enhanced expression of c-Myc protein, but without detectable c-Myc molecular alterations. FISH studies did not identify consistent etiologies of molecular dysregulation, and future studies with other molecular approaches may be required to elucidate the molecular pathogenesis of childhood T-LBL.

Smock, Kristi J.; Nelson, Marilu; Tripp, Sheryl R.; Sanger, Warren G.; Abromowitch, Minnie; Cairo, Mitchell S.; Perkins, Sherrie L.

2009-01-01

353

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

PubMed Central

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.

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

2013-01-01

354

Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora.  

PubMed

In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures. PMID:16935246

Sakai, Kenji; Ezaki, Yutaka

2006-06-01

355

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

Microsoft Academic Search

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

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

356

Amplified c-MYC sequences localized by fluorescence in-situ hybridization on double minute chromosomes in acute myeloid leukemias  

Microsoft Academic Search

Double minute chromosomes (dmin) are small acentric fragments frequently observed when karyotyping human tumor cells. They are considered the cytogenetic manifestation of gene amplification. The finding of dmin in leukemia is a rare event usually associated with progression of the disease and unfavorable prognosis. We present four patients affected by myeloid disorders with an abnormal karyotype and a variable number

Giuseppina Fugazza; Roberto Bruzzone; Laura Puppo; Franco Patrone; Mario Sessarego

1997-01-01

357

Recent advances in image analysis of chromosomes subjected to HG-banding and fluorescent in situ hybridization (FISH)  

Microsoft Academic Search

The use of cooled charge-coupled device (CCD) cameras and recently developed GIS software offer new opportunities for the analysis of chromosomes subjected to HG-banding. We evaluated the efficiency and sensitivity of CCD microscopy to the detection of the genomic origin of marker chromosome with partial trisomy in cells from the bone marrow of the patient with Myelodysplastic syndrome (MDS-RA). The

Angelina Novak; Aleksandar Jovanovic

1998-01-01

358

Immunohistochemistry and fluorescence in situ hybridization assessment of HER2 in clinical trials of adjuvant therapy for breast cancer (NCCTG N9831, BCIRG 006, and BCIRG 005).  

PubMed

A comprehensive, blinded, pathology evaluation of HER2 testing in HER2-positive/negative breast cancers was performed among three central laboratories. Immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) analyses were performed on 389 tumor blocks from three large adjuvant trials: N9831, BCIRG-006, and BCIRG-005. In 123 cases, multiple blocks were examined. HER2 status was defined according to FDA-approved guidelines and was independently re-assessed at each site. Discordant cases were adjudicated at an on-site, face-to-face meeting. Results across three independent pathologists were concordant by IHC in 351/381 (92 %) and FISH in 343/373 (92 %) blocks. Upon adjudication, consensus was reached on 16/30 and 18/30 of discordant IHC and FISH cases, respectively, resulting in overall concordance rates of 96 and 97 %. Among 155 HER2-negative blocks, HER2 status was confirmed in 153 (99 %). In the subset of 102 HER2-positive patients from N9831/BCIRG-006, primary blocks from discordant cases were selected, especially those with discordant test between local and central laboratories. HER2 status was confirmed in 73 (72 %) of these cases. Among 118 and 113 cases with IHC and FISH results and >1 block evaluable, block-to-block variability/heterogeneity in HER2 results was seen in 10 and 5 %, respectively. IHC-/FISH- was confirmed for 57/59 (97 %) primary blocks from N9831 (locally positive, but centrally negative); however, 5/22 (23 %) secondary blocks showed HER2 positivity. Among 53 N9831 patients with HER2-normal disease adjudicated as IHC-/FISH-(although locally positive), there was a non-statistically significant improvement in disease-free survival with concurrent trastuzumab compared to chemotherapy alone (adjusted hazard ratio 0.34; 95 % CI, 0.11-1.05; p = 0.06). There were similar agreements for IHC and FISH among pathologists (92 % each). Agreement was improved at adjudication (96 %). HER2 tumor heterogeneity appears to partially explain discordant results in cases initially tested as positive and subsequently called negative. PMID:23420271

Perez, Edith A; Press, Michael F; Dueck, Amylou C; Jenkins, Robert B; Kim, Chungyeul; Chen, Beiyun; Villalobos, Ivonne; Paik, Soonmyung; Buyse, Marc; Wiktor, Anne E; Meyer, Reid; Finnigan, Melanie; Zujewski, Joanne; Shing, Mona; Stern, Howard M; Lingle, Wilma L; Reinholz, Monica M; Slamon, Dennis J

2013-02-01

359

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

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.

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

360

Multicolor fluorescence in situ hybridization (M-FISH) on cells from urine for the detection of bladder cancer  

Microsoft Academic Search

Bladder cancer is the fifth most common cancer in adults. Because of the high recurrence rate (up to 70%) new tumor markers for urine are necessary for monitoring patients. In this study, we investigated the value of M-FISH on cells from urine for the detection of bladder cancer. Urine samples from 141 patients suspicious of bladder cancer were analyzed in

K. Junker; T. Fritsch; A. Hartmann; W. Schulze; J. Schubert

2006-01-01

361

Morphology assessment and fluorescence in situ hybridization of the same spermatozoon using a computerized cell-scanning system  

Microsoft Academic Search

BACKGROUND: Poor sperm morphology is statistically associated with an increase in the incidence of chromosome abnormalities. Our aim was to examine the possible correlation between chromosomal aberrations and sperm morphology in the same cell. METHODS: 12349 spermatozoa from 7 teratozoospermic and one globozoospermic patients, and from 3 fertile donors were analyzed using a system which scans for cell morphology and

D. Strassburger; M. Reichart; S. Kaufman; E. Kasterstein; D. Komarovsky; S. Friedler; M. Schachter; R. Ron-El; A. Raziel

2006-01-01

362

Assignment of the human dihydropyrimidine dehydrogenase gene (DPYD) to chromosome region 1p22 by fluorescence in situ hybridization  

SciTech Connect

Dihydropyrimidine dehydrogenase (DPD, EC 1.3.1.2) is the initial and rate-limiting enzyme in the three-step pathway of uracil and thymine catabolism leading to the formation of {beta}-alanine and {beta}-aminobutyric acid, respectively. Several studies have demonstrated the importance of DPD in cancer patients, particularly in those lacking or having only low levels of activity. Patients exhibiting severe toxicity when administered 5-fluorouracil were shown to have low DPD activity. Studies of affected families demonstrated that the deficiency was inherited in an autosomal recessive pattern. DPD deficiency is one of several inherited disorders of pyrimidine metabolism, clinically termed thymine-uracil-uria. 14 refs., 1 fig.

Takai, Setsuo [International Medical Center, Tokyo (Japan)] [International Medical Center, Tokyo (Japan); Fernandez-Salguero, Pedro; Kimura, Shioko [National Cancer Institute, Bethesda, MD (United States)] [and others] [National Cancer Institute, Bethesda, MD (United States); and others

1994-12-01

363

Miller-Dieker syndrome associated with duplication of 17p13.3 confirmed by fluorescence in situ hybridization (FISH)  

SciTech Connect

Miller-Dieker syndrome is characterized by profound mental retardation, craniofacial abnormalities, and lissencephaly (smooth brain). Microscopic or submicroscopic deletions of the 17p13.3 region have been reported in Miller-Dieker patients. We report a patient with this syndrome in whom a duplication of the 17p13.3 region was detected by FISH. The 9-year-old female proband was referred because of features of Miller-Dieker syndrome: microcephaly, profound psychomotor retardation, seizures, characteristic facies, and lissencephaly shown by MRI studies. High-resolution G-banding failed to demonstrate an abnormality in chromosome 17. However, FISH analysis with the DNA probe (Oncor No. 5101) specific for Miller-Dieker region of chromosome 17p13.3 demonstrated duplication of this segment instead of the classic deletion. We know of no other report of Miller-Dieker syndrome associated with duplication of 17p13.3. The family study revealed normal chromosomes in both parents by cytogenetic and FISH analysis. Our investigation suggests that duplications, as well as deletions, of the 17p13.3 region are associated with the Miller-Dieker syndrome. The presence of deletions or duplications of the same chromosomal region in patients with features of Miller-Dieker syndrome suggests that its pathogenesis may be due to gene dosage effects.

Li, S.; Tuck-Muller, C.M.; Martinez, J.E. [Univ. of South Alabama, Mobile, AL (United States)] [and others

1994-09-01

364

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

SciTech Connect

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.

Korenberg, J.R.

1997-12-31

365

Assignment of the human glypican gene (GPC1) to 2q35-q37 by fluorescence in situ hybridization  

SciTech Connect

Cell surface heparan sulfate proteoglycans are composed of a membrane-associated protein core substituted with a variable number of heparan sulfate chains. Two different cell surface heparan sulfate proteoglycan families can be distinguished: the syndecan-like integral membrane proteoglycans (SLIPS), with a core protein spanning the cytoplasmic membrane; and the glypican-related integral membrane proteoglycans (GRIPS), with a core protein anchored to the cytoplasmic membrane via a glycosyl phosphatidylinositol. The functions of these cell surface proteoglycans remain elusive, but because of their interactions with specific proteins, they are believed to be implicated in cell adhesion, cell growth, and control of proteolytic and lipolytic pathways. Glypican is the only human glypiated heparan sulfate proteoglycan that has so far been identified by cloning. Due to its highly conserved sequence, it has been speculated that this protein may support an essential function. Glypican is differentially expressed in different cell types and tissues. It is abundant in the adult rat brain, where it occurs in subpopulations of projection neurons. At this site, it may stabilize and activate neurotrophic polypeptide growth factors and mediate neuronal adhesion mechanisms that are essential for neuronal survival and injury responses. To learn more about glypican and to study its possible association with hereditary neuronal disorders, we have identified its chromosomal location. 6 refs., 1 fig.

Vermeesch, J.R.; Mertens, G.; David, G. [Univ. of Leuven (Belgium)] [and others] [Univ. of Leuven (Belgium); and others

1995-01-01

366

Localization of the human cytoplasmic dynein heavy chain (DNECL) to 14qter by fluorescence in situ hybridization  

SciTech Connect

Dyneins are a group of microtubule-activated ATPases that serve to convert chemical energy into mechanical energy. They have been divided into two large subgroups, namely the axonemal and cytoplasmic dyneins. Cytoplasmic dynein has been implicated in a variety of other forms of intracellular m