Fluorescence in situ hybridization and Y ring chromosome

Summary Investigations by fluorescence in situ hybridization and a Y-specific probe (Y190) of a male patient with a Y ring chromosome, 46,X,r(Y) showed four bright fluorescent spots within the ring. Thus, using this technique, it is possible to suggest that the ring originates from the duplication of the short arms of the Y chromosome.     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

Fluorescence in situ hybridization in paraffin tissue sections: pretreatment protocol

Fluorescence in situ hybridization has found wide application in the enumeration of gene and chromosome copy number both in isolated cells and in tissue sections. However, the technique has been less widely used than would be expected in formalin-fixed paraffin processed (archival) tissue. This article describes a method for assessing archival tissue sections, following pretreatment, before applying DNA probes, that gives consistent, reliable results.     

Fluorescence in situ hybridization in studying the human genome

Physical chromosome mapping by fluorescence in situ hybridization (FISH) is among the major lines of research on the human genome (as well as genomes of numerous other organisms). To localize particular genes or anonymous DNA sequences on individual chromosomes or chromosome regions, FISH was developed in the late 1980s and early 1990s, when the International Human Genome Project and the Russian program Human Genome were launched. Now FISH continues to play a prominent part in studies of the human genome. The review considers the major steps of FISH development in Russia with special emphasis on the key roles of the Institute of Cytology and Genetics (Novosibirsk) and Engelhardt Institute of Molecular Biology (Moscow). Physical mapping of human chromosomes 3 and 13 by FISH is described in detail. The promotion of FISH in Russia contributed to the progress in the related fields such as comparative animal genomics (ZOO-FISH) and studies of plant chromosomes.     

Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.     

Fluorescence in situ hybridization method for co-localization of mRNA and GEP

Co-localization studies using green fluorescent protein (GFP) and fluorescence immunohistochemistry have become commonplace. However, co-localization studies using GFP and mRNA in situ hybridization are rare, in large part because typical in situ hybridization reaction conditions often lead to the loss of GFP fluorescence. Here, we describe a new fluorescence mRNA in situ hybridization protocol using cRNA riboprobes that leaves GFP fluorescence intact. This protocol is based on a urea-based hybridization buffer and the Tyramide Signal Amplification system. This protocol should provide researchers engaged in the use of GFP with a solid starting point for adapting their own in situ hybridization protocols.     

Fluorescence in situ hybridization on vibratome sections of plant tissues

This protocol describes the application of fluorescence in situ hybridization (FISH) to three-dimensionally (3D) preserved tissue sections derived from intact plant structures such as roots or florets. The method is based on the combination of vibratome sectioning with confocal microscopy. The protocol provides an excellent tool to investigate chromosome organization in plant nuclei in all cell types and has been used on tissues of both monocot and dicot plant species. The visualization of 3D well-preserved tissues means that cell types can be confidently identified. For example, meiocytes can be clearly identified at all stages of meiosis and can be imaged in the context of their surrounding maternal tissue. FISH can be used to localize centromeres, telomeres, repetitive regions as well as unique regions, and total genomic DNAs can be used as probes to visualize chromosomes or chromosome segments. The method can be adapted to RNA FISH and can be combined with immunofluorescence labeling. Once the desired plant material is sectioned, which depends on the number of samples, the protocol that we present here can be carried out within 3 d.     

Fluorescence in situ hybridization of single copy transgenes in rice chromosomes

Summary Fluorescence in situ hybridization (FISH) is a powerful tool for visualizing the chromosomal location of targeted sequences and has been applied in many areas, including karyotyping, breeding and characterization of genes introduced into the plant genome. A simple, routine and sensitive FISH procedure was developed for localizing single copy genes in rice (Oryza sativa L.) metaphase chromosomes. We used digoxygenin-labeled endogenous or T-DNA sequences as small as 5.6 kb to probe corresponding endogenous sequences or the T-DNA insert in denatured rice metaphase chromosomes prepared from root meristem tissue. The hybridized probe sequence was labeled with cy3-conjugated anti-mouse IgG and visualized using fluorescence microscopy. Single copy and multiple copy introduced T-DNA sequences, as well as endogenous sequences, were localized on the chromosomes. The FISH protocol was effectively used to sereen the chromosomal location of introduced T-DNA and number of integration loci in rice.     

Fluorescence in situ hybridization (FISH)--application in research and diagnostics

The methods of molecular cytogenetics, in particular fluorescence in situ hybridization (FISH), are widely applied in cytogenetics for identification of numerical and structural chromosomal abnormalities, which are difficult to detect by routine cytogenetic techniques. Due to many advantages, FISH is used in research (gene mapping, gene expression studies, interspecies chromosome homology), and clinical diagnostics (chromosomal aberrations analysis in pre- and postnatal diagnostics, oncology). The techniques of in situ hybridization (ISH) are often employed in addition to classical banding techniques, in case where banding pattern is not reliable. This paper focuses on particular clinical examples, where FISH was successfully used to identify structural and numerical chromosomal aberrations.     

Fluorescence in situ hybridization of YAC clones after Alu-PCR amplification.

Alu-PCR protocols were optimized for the generation of human DNA probes from yeast strains containing yeast artificial chromosomes (YACs) with human inserts between 100 and 800 kb in size. The resulting DNA probes were used in chromosome in situ suppression (CISS) hybridization experiments. Strong fluorescent signals on both chromatids indicated the localization of specific YAC clones, while two clearly distinguishable signals were observed in greater than or equal to 90% of diploid nuclei. Signal intensities were generally comparable to those observed using chromosome-specific alphoid DNA probes. This approach will facilitate the rapid mapping of YAC clones and their use in chromosome analysis at all stages of the cell cycle.     

Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues

In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms.     

Hyperdiploidy and apparent aneusomy in mesothelial cells from non-malignant effusions as detected by fluorescence in situ hybridization (FISH)

Interphase cytogenetics by fluorescence in situ hybridization (FISH) can be used to detect malignant cells characterized by chromosomal aneuploidy. However, apparent aneusomy in normal "control" tissues has to be considered when using FISH as diagnostic tool. In effusions as model tissue exposed to metastasis, the definition of cut-off levels for background aneusomy by FISH was aimed in this study. Using centromeric probes representing chromosomes 7, 8, 11, 12, 17 and 18, extensive chromosome copy number enumeration by single-color FISH analysis was performed in pleural and ascitic effusions derived from 15 patients with various, non-malignant diseases. In all effusions, cells with gain of hybridization signals for several or all chromosomes tested were found (in up to 1.94% of cells). A consistent finding was high grade hyperdiploidy (>4 centromeric signals). Mesothelial elements mainly contributed to hyperdiploidy in effusions, as demonstrated by a combined analysis of FISH and immunocytochemistry with staining for cytokeratin. Dual-color FISH analysis showed that hyperdiploidy was predominantly corresponding to polyploidization; however, there were always minor cell populations classified as aneuploid by dual-color FISH. In conclusion, stringent criteria have to be applied to distinguish malignancy-related aneuploidy from background aneusomy by FISH.     

Fluorescence in situ hybridization (FISH) for direct visualization of microorganisms

As a technique allowing simultaneous visualization, identification, enumeration and localization of individual microbial cells, fluorescence in situ hybridization (FISH) is useful for many applications in all fields of microbiology. FISH not only allows the detection of culturable microorganisms, but also of yet-to-be cultured (so-called unculturable) organisms, and can therefore help in understanding complex microbial communities. In this review, methodological aspects, as well as problems and pitfalls of FISH are discussed in an examination of past, present and future applications.     

Fluorescence in situ hybridization analysis of the prokaryotic community inhabiting crystallizer ponds

A fluorescence in situ hybridization (FISH) protocol suitable for the identification of prokaryotes inhabiting hypersaline environments was developed and applied to several crystallizer ponds with salinities above 36% from a multipond solar saltern in Alicante, Spain. Two morphotypes were abundant in these environments: rods and square or square-like prokaryotes that could be affiliated to Bacteria and Archaea, respectively, by FISH with domain-specific probes. FISH with a newly designed probe proved that the archaeal 16S rDNA sequence most frequently recovered from the crystallizers, SPhT, originated from the dominant square-like prokaryotes. These uncultured prokaryotes have the morphology of Walsby's square bacteria. Additionally, FISH with a probe targeted to the genus Haloarcula , members of which are frequently isolated from this environment, indicated that this genus accounts for less than 0.1% of the total prokaryotic community.     

Fluorescence in situ hybridization to Y chromosomes in decondensed human sperm nuclei

Human sperm nuclei were isolated with mixed alkyltrimethylammonium bromide and dithiothreitol (MATAB/DTT) and decondensed by treatments with lithium diiodosalicylate (LIS), sodium chloride, or Tris salts. Concentrations as low as 1 mM LIS induced measurable nuclear swelling compared to 600 mM required for the other two salts. As measured by image analyses, the projected nuclear area increased linearly up to approximately fivefold with LIS concentrations up to 10 mM. Swollen nuclei also maintained the elliptical shapes characteristic of the human sperm head. Expanded sperm nuclei of three men were hybridized with a fluorescently labeled 3.4 kb Y chromosome-specific repetitive DNA probe; 50.1% of the nuclei of each semen sample showed fluorescent labeling over a part of the nucleus indicating presence of the Y chromosome. In comparison, unswollen sperm did not yield reliable hybridization signals. This procedure is suitable for determining the proportion of human sperm with Y chromosomes and can be used to evaluate sperm separation techniques. The availability of probes specific for most human chromosomes suggests that this procedure may find general application in studies of sperm chromosomal constitution.     

Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria

Fluorescence in situ hybridization (FISH) with horseradish peroxidase (HRP)-labeled oligonucleotide probes and tyramide signal amplification, also known as catalyzed reporter deposition (CARD), is currently not generally applicable to heterotrophic bacteria in marine samples. Penetration of the HRP molecule into bacterial cells requires permeabilization procedures that cause high and most probably species-selective cell loss. Here we present an improved protocol for CARD-FISH of marine planktonic and benthic microbial assemblages. After concentration of samples onto membrane filters and subsequent embedding of filters in low-gelling-point agarose, no decrease in bacterial cell numbers was observed during 90 min of lysozyme incubation (10 mg ml(-1) at 37 degrees C). The detection rates of coastal North Sea bacterioplankton by CARD-FISH with a general bacterial probe (EUB338-HRP) were significantly higher (mean, 94% of total cell counts; range, 85 to 100%) than that with a monolabeled probe (EUB338-mono; mean, 48%; range, 19 to 66%). Virtually no unspecific staining was observed after CARD-FISH with an antisense EUB338-HRP. Members of the marine SAR86 clade were undetectable by FISH with a monolabeled probe; however, a substantial population was visualized by CARD-FISH (mean, 7%; range, 3 to 13%). Detection rates of EUB338-HRP in Wadden Sea sediments (mean, 81%; range, 53 to 100%) were almost twice as high as the detection rates of EUB338-mono (mean, 44%; range, 25 to 71%). The enhanced fluorescence intensities and signal-to-background ratios make CARD-FISH superior to FISH with directly labeled oligonucleotides for the staining of bacteria with low rRNA content in the marine environment.     

Fluorescence in situ hybridization and spectral imaging analysis of human oocytes and first polar bodies.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35-39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.