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PubMedDate not supplied

Significant hybridization differences in comparative genomic hybridization due to nucleotides used for DNA labelling and to DNA chosen for cohybridization.

OBJECTIVE: Comparative genomic hybridization (CGH) has been established as an informative technique in genetic analysis. However, differences in the ratio of hybridization intensities were reported for particular chromosomes, which may affect CGH results. The aim of this study was to define these differences in more detail. For this purpose, CGH results of 70 samples of bone marrow cells (BMC) with normal karyotype in conventional cytogenetics (CC) were evaluated using seven different reference DNAs and two different DNA labeling systems. METHODS AND RESULTS: CGH using fluorochrome-conjugated nucleotides for DNA labeling indicated signal deviations in 21/70 BMC samples. Deviations affected chromosomes 1 (n = 21), 2 (n = 11), 4 (n = 11), 5 (n = 9), 6 (n = 7), 7 (n = 2), 8 (n = 2), 12 (n = 5), 13 (n = 15), 14 (n = 1), 16 (n = 17), 17 (n = 11), 19 (n = 21), 20 (n = 12), and/or 22 (n = 17). None of the imbalances were confirmed by fluorescence in situ hybridization (FISH). Using digoxigenin and biotin-conjugated nucleotides in exemplary cases (n = 5) led to the disappearance of the signal deviations. Repeated CGH experiments using seven different reference DNAs showed remarkable variations in the signal deviations. CONCLUSION: Hybridization differences depend not only on the hapten or fluorochrome-labeled nucleotides used for DNA labeling, but also on the reference DNA chosen. Therefore, close control of CGH experiments is mandatory, and additional techniques such as FISH should be performed to confirm the results obtained by CGH.

Adult↗

PubMed1990

Genomic in situ hybridization to sectioned nuclei shows chromosome domains in grass hybrids.

In situ hybridization using biotinylated total genomic DNA and avidin detection systems was adapted for examination of thin-sectioned plant material in the light and electron microscopes. Root tip material was preserved prior to sectioning, so that the in vivo disposition of the chromatin was maintained. Use of total genomic DNA from Secale africanum as a probe enabled the chromatin from the two parental genomes in the grass hybrid Hordeum chilense x S. africanum to be distinguished. The biotinylated probe preferentially labelled the chromosomes of S. africanum origin. DNA-DNA hybrids were visualized at the light-microscope level by Texas Red fluorescence and at the electron-microscope level by the enzymic precipitation of DAB (diaminobenzidine) or by colloidal gold particles. The use of thin sections allowed the location of probe hybridization to be established unequivocally in both metaphase and interphase nuclei. Analysis of interphase nuclei showed that chromatin originating from the two parental genomes did not intermix but occupied distinct domains.

Chromatin↗

PubMedDate not supplied

Chromosome arm 20q gains and other genomic alterations in esophageal squamous cell carcinoma, as analyzed by comparative genomic hybridization and fluorescence in situ hybridization.

BACKGROUND/AIMS: Our recent analysis of gastric cancers and colorectal cancers using comparative genomic hybridization revealed a novel, high frequent copy number increases the long arm of chromosome 20 in association with possible involvement of liver metastases and poor prognosis. This led to further comparative genomic hybridization analysis of chromosomal aberrations in primary tumors of esophageal squamous cell carcinoma. The aim of the study presented here was to analyze the chromosomal aberrations and to determine the numbers of copies of AIB1, BTAK, DcR3 and E2F1 as putative target genes on chromosome 20q as well as their expression and relation to clinicopathological features in 41 primary tumors of esophageal squamous cell carcinoma. METHODOLOGY: We used comparative genomic hybridization to screen 41 primary tumors of esophageal squamous cell carcinoma for changes in the number of copies of DNA sequences. To further characterize the gain of DNA sequences at 20q, we also performed fluorescence in situ hybridization analysis. We examined the relationship between these changes and clinicopathological factors. RESULTS: Gains in chromosome arm 20q were detected (34.1%) as well as a high level of gain in 20q12-13 (4.8%). AIB1 amplification was observed in 4.9% (2/41), BTAK amplification in 9.8% (4/41), DcR3 amplification was in 4.9% (2/41), and E2F1 amplification in 7.3% (3/41). The survival of patients with BTAK or E2F1 amplification was significantly lower than that of patients without these abnormalities. CONCLUSIONS: These findings provide evidence for a number of previously unknown genomic aberrations in esophageal squamous cell carcinoma, suggesting the existence of target regions relevant to its progression. Esophageal squamous cell carcinoma with 20q gain showed extensive lung metastases, pleural effusion and liver metastases and poorer prognosis compared to cases without 20q gain. Our results suggest that amplification of BTAK or E2F1 are likely to lead to an increase in the number of malignant phenotypes of esophageal squamous cell carcinoma and that these aberrations can be expected to be useful as markers of poor prognosis.

Aged↗

PubMed1995

A new hybrid cell line containing only human chromosome 15 selected through fluorescence in situ hybridization and characterized by Alu-PCR amplification of the human DNA.

Human Cot1 DNA directly labeled with dUTP-fluorochromes (FITC, Rhodamin) and used as a probe, allowed rapid detection of one or a few human chromosomes in human-hamster hybrid cells by in situ hybridization (FISH). A hybrid cell line containing only a human acrocentric chromosome was isolated (CH35B2D). The DNA from this hybrid was used for PCR amplification with a single Alu (A33) primer. After agarose gel electrophoresis, a continuum of intense bands of between 400-700 bp was observed with A33-PCR products. No amplified product was visible with hamster DNA. FISH on normal human metaphases of biotinylated Alu-PCR products obtained with Alu A33 and compétition with human Cot1 DNA showed decoration, with high specificity for chromosome 15. It was identified after R banding obtained with PI or DAPI in an antifade adjusted to pH11 with NaOH. Under the applied conditions, the Alu (A33) products are expected to be useful for characterization, by specific decoration of chromosome 15 aberrations in pathological cells. CH35B2D could be employed for functional studies of genes located on chromosome 15.

Animals↗

PubMed1993

Gene assignment by quantitative hybridization analysis of somatic cell hybrids.

Since the inception of somatic cell hybridization technology, the number of genes mapped to a particular chromosome or region of a chromosome has increased exponentially. Conventional assignment relies on the interpretation and designation of concordance to the various panel members. Assignment of genes to individual chromosomes may be ambiguous if the representation of the individual chromosomes within the hybrid panel is not considered. To overcome this inherent limitation, we have developed a computer-assisted method to assign genes to chromosomes. This assignment utilizes an integrative statistical analysis procedure to reconcile chromosomal representation of each member of the somatic cell hybrid panel. In this manner, the intensity of the corresponding bands appearing on the autoradiographic image reflects the prevalence of that specific gene-containing chromosome within the somatic cell hybrid. The statistical method described above provides the foundation for an independent means to assign genes to a specific chromosome. We have utilized this method to assign the human lysyl oxidase gene to chromosome 5.

Animals↗

PubMed1996

Enumeration of interphase chromosomes. Comparison of visual in situ hybridization and confocal fluorescence in situ hybridization.

OBJECTIVE: To compare two visual enumeration methods to determine whether the confocal approach yielded better counts of chromosome-specific hybridization sites. STUDY DESIGN: Brightfield microscopy was used to count in situ hybridization (ISH) sites in 4-microns tissue sections. Confocal microscopy was used to collect three-dimensional (3D) data sets from fluorescence in situ hybridization (FISH) preparations made with sections of various thicknesses. Analysis of the confocal images relied on custom-built interactive visualization software. RESULTS: The confocal method yielded higher average counts of hybridization sites per nucleus due to fewer truncated nuclei in thicker sections and to visual exclusion of the truncated nuclei that remained. Optimal section thickness was 8-12 microns. Limited penetration by FISH reagents restricted the use of thicker sections. CONCLUSION: Analysis of intact nuclei visualized in three dimensions was more sensitive in demonstrating high centromere number than was brightfield ISH analysis of 4-microns sections. Improvements in semiautomated interactive software may make the confocal approach practical for accurate evaluation of chromosome number in precise histologic contexts.

Carcinoma, Transitional Cell↗

PubMed1991

A rapid and economical method for hybrid purity testing of tomato (Lycopersicon esculentum L.) F1 hybrids using ultrathin-layer isoelectric focusing of alcohol dehydrogenase variants from seeds.

In the cultivated tomato (Lycopersicon esculentum L.), two electrophoretic variants of the enzyme alcohol dehydrogenase (ADH) encoded by the alleles Adh-1+ and Adh-1(1) are found. A rapid and economic method for testing the hybrid purity of tomato F1 seeds, based on the expected presence of Adh-1 alleles, was developed. The method is based on the analysis of the ADH variants by ultrathin-layer isoelectric focusing, pH range 3-10, of crude extracts from imbibed seeds, followed by enzyme activity staining. The isoelectric points (pI's) of the ADH variants were estimated to be 5.5 and 5.7 for Adh-1+ and Adh-1(1), respectively. Using the procedure described and a newly developed sample applicator strip, it is possible for one person to routinely analyze 1152 seeds per day using only a single electrophoresis unit. An investigation of a large number of inbred lines, both experimental and commercial hybrids, together with open-pollinated varieties, showed the potential of the method. Among F1 hybrids, a higher frequency of the Adh-1(1) allele was found than among open pollinated varieties, suggesting that F1 hybrid breeding has resulted in a higher frequency of Adh-1(1) alleles by selection of linked genes.

Alcohol Dehydrogenase↗

PubMed1993

Cytogenetic profiling using fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH).

Fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) allow cytogenetic analyses of primary tumors without culture. CGH allows detection and mapping of allelic imbalance by simultaneous in situ hybridization of differentially labeled tumor (green fluorescing) and normal DNA (red fluorescing) to a normal human metaphase spread. Regions of increased or decreased copy number in the tumor are mapped onto the normal metaphase chromosomes as increases or decreases in the green to red fluorescence ratio. This technique gives a comprehensive assessment of gene dosage imbalance throughout the tumor. However, it is limited, at present, to fairly large tumors containing few normal cells. FISH, on the other hand, allows analysis of DNA sequence copy number at specific loci in single nuclei. A wide variety of DNA probes is available for FISH, including chromosome-specific probes which hybridize to alpha-satellite pericentromeric DNA regions (to detect changes in specific chromosome copy number and overall ploidy) and specific locus probes targeting 20-150 kilobase sequences (to detect specific amplifications, deletions, breakpoints, or rearrangements). FISH using these probes has been applied to interphase nuclei in touch preparations, smears from fine needle aspirates, and thin (< 6 microns) and thick (> 20 microns) sections cut from formalin-fixed, paraffin-embedded tissue. Analysis of thick sections allows accurate actual signal enumeration within the histological context. This approach may allow analysis of subtle premalignant, early malignant, and infiltrating tumors in which malignant cells must be differentiated from nonmalignant cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Chromosome Aberrations↗

PubMed1991

Investigation of a quantitative post-hybridization signal amplification system for mRNA-oligodeoxyribonucleotide in situ hybridization.

This study was undertaken to assess a post-hybridization amplification system for increasing the sensitivity of in-situ hybridization with oligodeoxyribonucleotide (oligonucleotide) probes. The technique employs a multilayer streptavidin-biotinylated link protein amplification technique, similar to that used in the ABV histochemical amplification system, to attach increasing numbers of radiolabelled streptavidin molecules to a biotinylated ologonucleotide probe. In tissue sections the amplification technique increases the signal-to-noise ratio dramatically, increasing the sensitivity of in situ hybridization and reducing the autoradiographic exposure time. On an artificial medium the amplification technique has been shown to increase the hybridization signal 100-fold when compared with a directly radiolabelled oligonucleotide probe. The technique could be used to compare relative amounts of target molecule, there being a linear relationship between the detectable signal and the log of the concentration of the target molecule.

Autoradiography↗

PubMed1993

Comparison of Virapap filter hybridization with polymerase chain reaction and Southern blot hybridization methods for detection of human papillomavirus in tonsillar and pharyngeal cancers.

A method using a commercial dot filter hybridization kit, Virapap, was compared with Southern blot hybridization and polymerase chain reaction (PCR) for the detection of human papillomavirus (HPV) types 16 and 18 in pharyngeal and tonsillar cancers of 12 patients as well as tonsillar biopsies from 28 patients with chronic tonsillitis. Concordant results between Virapap and PCR, Virapap and Southern hybridization, and PCR and Southern hybridization methods were obtained respectively in 41.7%, 58.3% and 83.3% of the cancer cases, and 67.9%, 67.9% and 85.7% of the control (tonsillitis) cases. Virapap false-positive results were found in 5 cancer cases and 5 control cases. Although the Virapap method is reported to be useful for detecting HPV in gynecological tissues, this method cannot be recommended for the detection of HPV in pharyngeal and tonsillar cancers.

Adolescent↗

PubMed1993

Cytogenetical localization of Zygotic hybrid rescue (Zhr), a Drosophila melanogaster gene that rescues interspecific hybrids from embryonic lethality.

Hybrid females from crosses between Drsophila melanogaster males and females of its sibling species, D. simulans, D. mauritiana, or D. sechellia die as embryos. This lethality is believed to be caused by incompatibility between the X chromosome of D. melanogaster and the maternal cytoplasm. Zygotic hybrid rescue (Zhr) prevents this embryonic lethality and has been cytogenetically mapped to a proximal region of the X chromosome of D. melanogaster, probably in the centromeric heterochromatin. We have carried out high resolution cytological mapping of Zhr using deficiencies and duplications of the X heterochromatin. Deletions of the Zhr+ gene from the hybrid genome exhibit the Zhr phenotype. On the contrary, addition of the wild-type gene to the hybrid genome causes embryonic lethality, regardless of sex. The Zhr locus has been narrowed down to the region covered by Dp(1;f)1162 but not covered Dp(1;f)1205, a chromosome carrying a duplication of heterochromatin located slightly distal to the In(1)sc8 heterochromatic break-point.

Animals↗

PubMed1994

A Lactuca universal hybridizer, and its use in creation of fertile interspecific somatic hybrids.

A Lactuca sativa cv. Ardente line heterozygous for a gene encoding resistance to kanamycin, a positive and dominant trait, was crossed with cv. Girelle, which is heterozygous for a recessive albinism marker. The resulting seeds yielded 25% albino seedlings, of which 50% were also resistant to kanamycin. Such plantlets (KR, a) grown in vitro were used for preparation of universal hybridizer protoplasts, since green buds that can develop on kanamycin containing-medium should result from fusion with any wild-type protoplast. To test the practicability of this selection scheme, we fused L. sativa KR, a protoplasts with protoplasts derived from various wild Lactuca as well as various other related species. Protoplast-derived cell colonies were selected for resistance to kanamycin at the regeneration stage. Green buds were regenerated after fusion with protoplasts of L. tatarica and of L. perennis. So far, 9 interspecific hybrid plants have been characterized morphologically. In addition, random amplified polymorphic DNA (RAPD) analysis with selected primers confirmed that these plants are indeed interspecific hybrids. Some plants are female-fertile and production of backcross progenies with L. sativa is in progress. Since many desirable traits such as resistances to viruses, bacteria and fungi (Bremia lactucae) have been characterized in wild Lactuca species, the use of somatic hybridization in breeding programmes now appears a practical possibility.

Base Sequence↗

PubMed1999

Progress and variations in two-hybrid and three-hybrid technologies.

The original yeast two-hybrid system and its variants have proven to be effective tools for identification and analysis of protein-protein, protein-DNA and protein-RNA interactions. The two-hybrid assay is being applied to the entire complement of proteins of the yeast Saccharomyces cerevisiae to characterize the network of protein-protein interactions in the eukaryotic cell. The development of nontranscriptional cytosolic and membrane-associated two-hybrid methods has made it possible to detect and examine a number of protein-protein interactions in their normal cellular locations. Small-molecule hybrid systems have been developed which can be used to study protein-ligand interactions and to activate cellular processes by forcing protein associations.

Enzymes↗

PubMedDate not supplied

Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

Fluorometry↗

PubMed2001

Use of DNA markers in prediction of hybrid performance and heterosis for a three-line hybrid system in rice.

Two Cytoplasmic Male Sterile lines were crossed with fourteen restorer lines of rice widely grown in the western regions of Maharashtra, India, to produce 28 F1 hybrids which were evaluated for eight agronomically important traits, contributing to yield potential, in replicated field trials. The hybrid performance was recorded along with heterosis and heterobeltiosis. All the rice lines under investigation were subjected to marker-based variability analysis. An attempt was made to correlate genetic distance based on specific markers for each trait individually, as well as average genetic distance based on all specific markers, with hybrid performance and heterosis, by regression analysis. Specific markers could cluster the parental lines in different groups and showed significant correlation with hybrid performance. The data also supports the proposition that epistasis is the basis of heterosis. The analysis, however, revealed a lack of significant predictive values for field application.

Cluster Analysis↗

PubMed1974

Expression of differentiated functions in hepatoma cell hybrids: high frequency of induction of mouse albumin production in rat hepatoma-mouse lymphoblast hybrids.

We have studied the production of serum albumin by somatic hybrids between well-differentiated 2s and 1s rat hepatoma cells (Faza), which produce serum albumin, and sub-diploid mouse leukemic lymphoblasts (Lc), which do not produce albumin. We determined the rat or mouse origin of the albumin by double immunodiffusion, using immuno-adsorbed noncrossreacting antisera. Each of 12 karyologically identified 2s hybrid clones (Lc2F) produces both rat and mouse albumin. Moreover, unlike 1s hybrids reported previously, eight of nine 1s hybrids (LcF) also produce mouse albumin; six of them produce rat albumin as well. One clone from the 1s cross produces only rat albumin.

Albumins↗

PubMed1996

Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.

Macromolecular interactions define many biological phenomena. Although genetic methods are available to identify novel protein-protein and DNA-protein interactions, no genetic system has thus far been described to identify molecules or mutations that dissociate known interactions. Herein, we describe genetic systems that detect such events in the yeast Saccharomyces cerevisiae. We have engineered yeast strains in which the interaction of two proteins expressed in the context of the two-hybrid system or the interaction between a DNA-binding protein and its binding site in the context of the one-hybrid system is deleterious to growth. Under these conditions, dissociation of the interaction provides a selective growth advantage, thereby facilitating detection. These methods referred to as the "reverse two-hybrid system" and "reverse one-hybrid system" facilitate the study of the structure-function relationships and regulation of protein-protein and DNA-protein interactions. They should also facilitate the selection of dissociator molecules that could be used as therapeutic agents.

Base Sequence↗

PubMed2001

Value of morphological criteria in diagnosing cervical HPV lesions confirmed by in situ hybridization and hybrid capture assay.

The present study evaluated the value of morphological criteria (binucleation, multinucleation, koilocytosis, spindle koilocytes, abnormal mitosis and dyskeratosis) in the diagnosis of cervical human papillomavirus (HPV) lesions confirmed by in situ hybridization (ISH) and hybrid capture (HC) assay. Colposcopic punch biopsies from a series of 138 women with abnormal Pap smears were examined on light microscopy and in situ hybridization (DAKO widespectrum cocktail probe) for HPV-induced morphological changes and HPV DNA, respectively. Cervical swabs were analyzed for HPV DNA of the oncogenic types using Hybrid Capture. CIN 2 and CIN 3 were found in 44 biopsies, CIN 1 in 62, and no evidence of HPV in 32 cases. HPV was detected by ISH in 51/138 (37%) cases and by HC in 66/138 (48%) lesions. With both tests, HPV DNA detection increased parallel with lesion severity, up to 70% and 59% in CIN 2/3 by HC and ISH, respectively OR 4.6 (1.7-12.1) and 10.1 (3.0-33.8). Among the histological criteria, multinucleation, binucleation and abnormal mitoses were significantly associated with HPV DNA detection. Multinucleation proved to be the strongest predictor of HPV DNA-positivity. Binucleation, abnormal mitosis, koilocytosis and spindle koilocytes were also reliable criteria of HPV lesions. Minor nuclear atypia, and "mild koilocytosis" were of no value in making this diagnosis.

Biopsy↗