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Results for “Hybridization”

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PubMed1982

Expression of the ASV src gene in hybrids between normal and virally transformed cells: specific suppression occurs in some hybrids but not others.

Somatic cell hybrids have been made between clones of rat cells transformed by avian sarcoma virus and rat or mouse cells that are untransformed. Intraspecies hybrids were either predominantly morphologically normal or predominantly transformed, some clones that formed transformed intraspecies hybrids yielding normal interspecies hybrids. Untransformed hybrids usually showed no detectable alteration in the structure or location of the integrated provirus, but viral RNA and pp60src kinase activities were much reduced. No decrease in viral gene expression was seen in transformed hybrids. Thus hybrid suppression of viral transformation, mediated in trans by the untransformed parent, is a specific event that depends on both untransformed and transformed parental parameters.

Animals↗

PubMed1995

Cloning mammary cell cDNAs from 17q12-q23 using interspecific somatic cell hybrids and subtractive hybridization.

We have cloned human genes that are encoded in the region 17q12-q23 and expressed in breast tissue using interspecific somatic cell hybrids and subtractive hybridization. Two mouse microcell hybrids containing fragments of human chromosome 17 with a nonoverlap region at 17q12-q23 were generated by microcell transfer. Radiolabeled cDNA was synthesized from the hybrid cell containing the 17q12-q23 interval and was subtracted with an excess of RNA from the hybrid cell lacking the interval. Resulting cDNA probes enriched for sequences from 17q12-q23 were used to screen a human premenopausal breast cDNA library, and 60 cDNAs were identified. Three of these cDNAs mapped to the hybrid cell nonoverlap region. These cDNAs were expressed in mammary epithelial cell hybrids, although none appeared to be breast-specific. Sequence analysis of the cDNAs revealed that clone 93A represents a previously unidentified gene, clone 98C has homology to an expressed sequence tag from goat mammary tissue, and clone 200A is identical to the human homologue of the Drosophila melanogaster flightless-I gene. These genes map outside a 1-cM region linked to early onset familial breast cancer but may be useful genetic markers in the 17q12-q23 region.

Animals↗

PubMed1989

Solution hybridization and enzyme immunoassay for biotinylated DNA-RNA hybrids to detect enteroviral RNA in cell culture.

A non-isotopic hybridization assay is described for detection of enteroviral RNA in cell culture. Two biotin-labelled cDNA probes, corresponding to 1 kb from the 5' end and 3.5 kb from the 3' end of the coxsackievirus B3 genome, were hybridized in solution with protease and detergent-treated cell culture suspensions. Labelled DNA-RNA hybrids were captured on microtiter plates coated with anti-biotin antibody and bound hybrids were measured with a beta-galactosidase-labelled monoclonal antibody specific for DNA-RNA hybrids. Coxsackie B3 was detected at a concentration of 500 pfu ml-1. The limit of detection for other enteroviruses ranged from 10(3.3) to 10(5.8) pfu ml-1. The enteroviruses that could be detected included coxsackie B1 and 3, coxsackie A1-6 and 15, poliovirus types 1-3, and enteroviruses 7, 11, and 71. ECHO 22 was the only enterovirus, of those that were tested, that could not be detected. The solution hybridization reaction and enzyme immunoassay for DNA-RNA hybrids does not require the use of radiolabelled probes or extraction of RNA with phenol. The assay yields a quantitative endpoint, which avoids the subjectivity inherent in membrane-based methods. These features would make the assay more adaptable to clinical laboratories than other formats which have been devised for measurement of viral RNA.

Animals↗

PubMed1992

Thermal degradation of RNA-RNA hybrids during hybridization in solution.

Degradation of single-stranded RNA molecules at high temperatures was examined in relation to the kinetics of RNA-RNA hybridization in solution. Eleven species (ranging from 670 bases to 3300 bases) of single-stranded RNAs transcribed from rotavirus genomic RNAs degraded significantly after 16 h of incubation at 65 degrees C. The hybridization of these 11 RNA molecules to the corresponding genomic RNAs, however, was completed within 30 min of incubation. Partially homologous hybrids that were once formed at an early time of incubation gradually degraded in proportion to the length of incubation at 65 degrees C. Thus, the length of hybridization has a critical effect on the final hybridization results. Furthermore, thermal hydrolysis of single-stranded RNA molecules provides a plausible explanation why the percent of nucleotide sequence mismatch allowed to form a stable hybrid in the RNA-RNA hybridization assays for rotavirus genes is much less than that predicted by calculation.

Base Composition↗

PubMed2003

Inheritance patterns of phenolics in F1, F2, and back-cross hybrids of willows: implications for herbivore responses to hybrid plants.

The aim of this study was to determine the inheritance pattern of phenolic secondary compounds in pure and hybrid willows and its consequences for plant resistance to leaf-feeding insects. F1, F2, and back-cross hybrids along with pure species were produced by hand pollination of pure, naturally-growing Salix caprea (L., Salicaceae) and S. repens (L.) plants. Leaf concentrations of condensed tannins and seven different phenolic glucosides were determined by using butanol-HCI and HPLC analyses. Insect herbivore leaf damage was measured on the same leaves as used for chemical analyses. We found hybrids to be approximately intermediate between the parental species: S. caprea with high levels of condensed tannins and no phenolic glucosides. and S. repens with low levels of condensed tannins and high levels of phenolic glucosides. We also found a negative correlation between concentrations of condensed tannins and phenolic glucosides, suggesting a trade-off in production of these two substances. F2 hybrids and the hybrid back-crossed to S. caprea were significantly more damaged by insect herbivores than the parental species and the F1 hybrid, indicating reduced resistance and possibly a selective disadvantage for these hybrid categories.

Adaptation, Physiological↗

PubMed2006

Kinetics of hybridization on surface oligonucleotide microchips: theory, experiment, and comparison with hybridization on gel-based microchips.

The optimal design of oligonucleotide microchips and efficient discrimination between perfect and mismatch duplexes strongly depend on the external transport of target DNA to the cells with immobilized probes as well as on respective association and dissociation rates at the duplex formation. In this paper we present the relevant theory for hybridization of DNA fragments with oligonucleotide probes immobilized in the cells on flat substrate. With minor modifications, our theory also is applicable to reaction-diffusion hybridization kinetics for the probes immobilized on the surface of microbeads immersed in hybridization solution. The main theoretical predictions are verified with control experiments. Besides that, we compared the characteristics of the surface and gel-based oligonucleotide microchips. The comparison was performed for the chips printed with the same pin robot, for the signals measured with the same devices and processed by the same technique, and for the same hybridization conditions. The sets of probe oligonucleotides and the concentrations of probes in respective solutions used for immobilization on each platform were identical as well. We found that, despite the slower hybridization kinetics, the fluorescence signals and mutation discrimination efficiency appeared to be higher for the gel-based microchips with respect to their surface counterparts even for the relatively short hybridization time about 0.5-1 hour. Both the divergence between signals for perfects and the difference in mutation discrimination efficiency for the counterpart platforms rapidly grow with incubation time. In particular, for hybridization during 3 h the signals for gel-based microchips surpassed their surface counterparts in 5-20 times, while the ratios of signals for perfect-mismatch pairs for gel microchips exceeded the corresponding ratios for surface microchips in 2-4 times. These effects may be attributed to the better immobilization efficiency and to the higher thermodynamic association constants for duplex formation within gel pads.

Electrophoresis↗

PubMed1993

Hybrid lethal systems in the Drosophila melanogaster species complex. I. The maternal hybrid rescue (mhr) gene of Drosophila simulans.

Hybrid females from Drosophila simulans females x Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as late larvae. The other two classes are sterile adults. Letting C, X, and Y designate egg cytoplasm, X, and Y chromosomes, respectively, and subscripts m and s stand for melanogaster and simulans, CmXmYs males are lethal in the larval stage and are rescued by the previously reported genes, Lhr (Lethal hybrid rescue) in simulans or Hmr (Hybrid male rescue) in melanogaster. We report here another rescue gene located on the second chromosome of simulans, mhr (maternal hybrid rescue) that, when present in the mother, rescues CsXmXs females from embryonic lethality. It has been postulated that the hybrids not carrying the Xs like CmXmYs males are larval lethal and that the hybrids carrying both the Cs and the Xm like CsXmXs females are embryonic lethal. According to these postulates CsXmYs males (obtained by mating attached-X simulans females to melanogaster males) should be doubly lethal, at both embryo and larval stages. When both rescuing genes are present, Hmr in the father and mhr in the mother, males of this genotype are fully viable, as predicted.

Animals↗

PubMed1992

Effect of the higher-order structure of tRNAs on the stability of hybrids with oligodeoxyribonucleotides: separation of tRNA by an efficient solution hybridization.

In the course of developing a method to purify a single tRNA species efficiently, we have examined hybridization efficiencies between some tRNAs and short oligodeoxyribonucleotide probes both by the filter and solution hybridization methods without denaturants. The hybridization efficiencies varied considerably among probes which are complementary to different regions of the tRNAs, although there was little efficiency variation in the probes toward DNA substrates including the same nucleotide sequence. This efficiency variation was shown to be due to tRNA-specific higher-order structures as well as a hypermodified nucleotide in the anticodon loop. Characterization of the tRNA-probe hybrids by both nondenaturing gel electrophoresis and chemical modification showed the existence of two stable hybridizing states as a function of ionic strength. Our results indicate that RNA molecules with a number of intramolecular base pairings are able to form stable hybrids with complementary sequences under nondenaturing conditions. On the basis of these data, an appropriate probe was designed to successfully purify yeast tRNA(Phe) by making a tRNA(Phe)-probe hybrid, which has a longer retention time in hydroxyapatite high performance liquid chromatography than the tRNA(Phe) itself.

Animals↗

PubMed1991

Evaluation of hybridization characteristics of a cloned pRF106 probe for Listeria monocytogenes detection and development of a nonisotopic colony hybridization assay.

An internal fragment (pRF106 fragment, ca. 500 bp) of a gene (msp) coding for a 60-kDa protein of Listeria monocytogenes serotype 1/2a was used to develop a screening method to discriminate between L. monocytogenes and avirulent Listeria spp. on primary isolation plates. The L. monocytogenes-derived probe fragment of pRF106 hybridized to a 13-kb fragment of L. monocytogenes and a 3-kb fragment of one cheese isolate strain of Listeria seeligeri under stringent hybridization conditions (mean thermal denaturation temperature [Tm]-5 degrees C). The probe also hybridized to a 6-kb fragment of Listeria innocua, Listeria ivanovii, and L. seeligeri under less stringent hybridization conditions (Tm-17 degrees C). The pRF106 fragment was labeled with digoxigenin-11-dUTP and used to develop a colony hybridization assay. Colonies from lithium chloride-phenylethanol-moxalactam agar were blotted onto nylon membranes. The cells were pretreated with microwaves before lysis with sodium hydroxide. DNA-DNA hybridization and posthybridization washing were done at high stringency (Tm-7 degrees C). The nonisotopic colony hybridization procedure was specific for L. monocytogenes when evaluated against pure cultures of L. monocytogenes and other Listeria species, excluding the cheese isolate of L. seeligeri. Also, it was specific for L. monocytogenes when evaluated with Listeria-negative food enrichment cultures that were inoculated in the laboratory with Listeria species.

DNA Probes↗

PubMed1980

Analysis of the cell adhesion mechanism using somatic cell hybrids. I. Aggregation of hybrid cells between adhesive V79 and non-adhesive Ehrlich's ascites tumour cells.

V79 Chinese hamster cells dissociated with 1 nm EDTA retain 2 kinds of cell adhesion mechanisms, one dependent on Ca2+ and the other independent of Ca2+. Ehrlich's ascites tumour (EAT) cells are provided with neither Ca2+-dependent nor CA2+-independent mechanisms. Studies on the aggregation of cells of 25 different hybrid clones obtained by fusing these 2 lined cells revealed the following points with regard to adhesive properties of hybrid cells. (1) The activity of the Ca2+-independent mechanism was lower in most hybrid clones than in parental V79 cells. (2) There were a few hybrid clones whose Ca2+-dependent mechanism activity was lower than in V79 cells. In these clones, the Ca2+-independent mechanism was also less active than the parental cells. (3) A hybrid clone with reduced Ca2+-dependent mechanism activity only was not found. (4) All the hybrid clones have at least one set of chromosomes derived from V79. (5) The number of chromosomes derived from EAT cells tended to be less in hybrid clones with lower aggregative ability. These results can be explained by assuming that the activity of the Ca2+-independent and Ca2+-dependent mechanisms of V79 cells may be differently inhibited by genes contained in EAT cells.

Animals↗

PubMed1985

Cell hybridization and cell agglutination. II. Enhancement of cell hybridization by polycations.

An efficient technique for hybridization of mammalian cells was developed by combining agglutination by pretreatment with polycations, such as polyarginine, and conventional polyethylene glycol(PEG)-mediated cell fusion. Polyarginine and subsequent PEG treatment resulted in markedly decreased viability in the treated cells, but addition of polyvinyl pyrrolidone or glycerol to the polyarginine prevented this cytotoxicity. Polyarginine was much more effective than polylysine or polyornithine in inducing hybridization. Other polycations, including polybrene and protamine but not DEAE-dextran, were also active in inducing hybridization. The condition of the cells at the time of polycation treatment was an important factor in the enhancement of hybridization. The condition of the cells at the time of polycation treatment was an important factor in the enhancement of hybridization. The enhancement of hybridization of cells in monolayer incubated for 2 h was much higher than that of cells incubated for 24 h. These findings suggest that polycations do not necessarily operate by agglutinating cells. The mechanism of polycation-enhanced cell hybridization is discussed.

Agglutination↗

PubMed2000

[Fluorescence in situ hybridization applied to the meiotic analysis and spontaneous chromosome translocation in the pollen mother cells of hybrids of Triticum-Haynaldia].

Fluorescence in situ hybridization was applied to the meiotic analysis and chromosome pairing in the pollen mother cells of hybrids of Triticum-Haynaldia. The results indicated that most (90.5%-93.2%) of the PMCs of two Triticum-Haynaldia hybrids contained two univalents involving one Haynaldia chromosome at metaphase I, and only 1 of 320 PMCs with homologous pairing between one wheat chromosome and one Haynaldia chromosome was observed in the hybrid of Guinong 22 x common wheat Chinese Spring, the frequency was 0.3%. The frequencies of the PMCs with mis-dividing chromosome ranged from 32.7% to 37.5% at anaphase I and from 20.5% to 24.4% at anaphase II in the hybrids between Triticum-Haynaldia. The frequency of Haynaldia chromatin translocating into Triticum chromosome was about 0.7% at anaphase I in the hybrid of Guinong 22 x common wheat Chinese Spring and was 0.8%-1.7% at anaphase II in two wheat-Haynaldia hybrids. Otherwise, the chromosome morphology involving Haynaldia chromatin was observed at preleptotene I, and the frequencies of chromatin nucleus at tetrad stage and at microspore stage were investigated. The time of chromosome translocation in meiosis and the transmitting frequencies of Haynaldia chromosome in wheat background were discussed.

Gene Frequency↗

PubMed1983

Suppression of tumorigenicity in somatic cell hybrids. II. Human chromosomes implicated as suppressors of tumorigenicity in hybrids with Chinese hamster ovary cells.

Nontumorigenic diploid human cells were fused with tumorigenic Chinese hamster ovary cells (CHO), and the hybrids were tested for tumorigenicity to determine if specific human chromosomes are associated with suppression of tumorigenicity in cell hybrids. Chromosome complements of cells of 62 nontumorigenic and 45 tumorigenic hybrids (divided into those of low, medium, and high tumorigenicity) as well as 44 tumors derived from the tumorigenic hybrids were determined by both analysis of banded chromosomes and assays of gene markers. Although no single human chromosome was consistently associated with the suppressed phenotype, chromosome 2 was never found in tumor cells, and chromosomes 9, 10, 11, and 17 were found at very low incidences in tumor cells, which suggested that they carry tumorigenicity suppressor information. Since not all suppressed hybrids contained these chromosomes, it is likely that they suppressed tumorigenicity only in combination with each other or other chromosomes. Nine chromosomes in 12 pairwise combinations of nonhomologous chromosomes were not found in tumor cells and were found at an incidence of 5% or less in hybrids of both medium and high tumorigenicity. Other experiments implicated 11 of these combinations involving only 8 chromosomes (chromosomes 4, 7, 8, 9, 10, 11, 13, and 17) as those primarily involved in suppression. Whether chromosome 2 requires another chromosome to effect suppression could not be determined. Further evaluations of the implicated suppressors, including selection of tumorigenic segregants from a panel of suppressed hybrids, again implicated the same chromosomes and their combinations in suppression. Oncogenes have been mapped to many of these chromosomes, and they are frequently involved in tumor-type-specific numerical or structural abnormalities in human neoplasias. The combined evidence suggests that specific human chromosomes of a normal cell carry genes that can regulate several cell phenotypes necessary for the expression of tumorigenicity.

Animals↗

PubMedDate not supplied

Tissue subtraction DNA in situ hybridization (TSDISH): in situ detection of DNA abnormalities in formalin-fixed paraffin-embedded tissue sections by subtraction hybridization with whole genomic DNA.

We have developed a novel in situ histochemical method of screening for genetic alterations of human malignancies by subtraction hybridization of genomic DNA without employing specific probes to give a colorimetric reaction. We identified a t(13;14) chromosome abnormality in the chromosome spread of a patient with multiple myeloma. In situ hybridization of a whole cell preparation of MCF-7 cell demonstrated reaction products as intranuclear dots-in all MCF-7 cells. We subsequently examined the cells of different foci of esophageal squamous cell carcinoma(10) and esophageal biopsy specimens (9) by this method. Hybridization with genomic DNA from the patients demonstrated no reaction products in the stromal cells of the esophagus. However, hybridization with reference DNA from a healthy individual demonstrated intranuclear reaction products in the stromal cells, possibly due to individual genomic differences. There were more intranuclear reaction products in the carcinoma cells than in the stromal cells when hybridized with reference DNA. When hybridized with the reference DNA above, the cells of the non-pathologic epithelium of 8 of 10 malignant esophagi demonstrated significantly more reaction product than the stromal cells of the some specimens. This was not detected in the cells of normal epithelium obtained from non-cancerous esophagi suggesting the accumulation of genetic alterations of the non-malignant epithelium of the cancerous esophagus. This method is thought to detect DNA alterations, including those which have not been previously identified, using genomic DNA for hybridization, and the results can be correlated with the morphological findings. Application of this in situ method, together with other molecular genetic techniques may contribute to the analysis of various genetic alterations of human malignancies using archival material.

Chromosome Aberrations↗

PubMed1999

Deletion of p16INK4A/CDKN2 and p15INK4B in human somatic cell hybrids and hybrid-derived tumors.

Deletion or epigenetic inactivation of the tumor suppressor gene p16INK4/CDKN2 (p16) has been observed in multiple human tumors. We assayed hybrid cell lines between human diploid fibroblasts and fibrosarcoma cells for p16 allelic status and expression and found that p16 was expressed in the parental diploid fibroblast cell lines used, whereas the parental fibrosarcoma cell line HT1080.6TG exhibited homozygous deletion of p16. Most immortalized hybrid cell lines derived from these parent cell lines, whether tumorigenic or nontumorigenic, exhibited loss of fibroblast-derived p16 alleles. All p16-negative hybrid cell lines also exhibited deletion of p15INK4B (p15). Hybrid cell lines yielded tumors upon s.c. injection into athymic nude mice regardless of p16/p15 status. Tumors derived from six p16/p15-positive hybrid cells, however, revealed deletions of both p16 and p15. When human diploid fibroblasts were fused with A388.6TG squamous cell carcinoma cells, which exhibit aberrant methylation of p16, the resulting hybrids again exhibited deletion of the unmethylated fibroblast-derived p16 alleles. Transfection of both HT1080.6TG and A388.6TG cells with wild-type p16 expression vector resulted in decreased clonogenicity in culture. Although the determinants directing genetic versus epigenetic inactivation of p16 and p15 remain unclear, these results demonstrate that p16-mediated growth suppression could be abrogated by either mechanism in somatic cell hybrids.

Animals↗

PubMed1996

Cloning Expressed cDNAS from Defined Chromosomal Regions Using Interspecific Microcell Hybrids and Subtractive Hybridization

Genes expressed from a defined genomic interval can be cloned using interspecific microcell hybrids and subtractive hybridization. This approach involves identifying a pair of interspecific hybrids that contain nearly isogenic fragments of a relevant chromosome, except for a region of nonoverlap encompassing the locus of interest. Radiolabeled cDNAs are synthesized from the hybrid cell line that contains the region of interest and hybridized in solution with an excess of biotinylated mRNA from the microcell hybrid lacking the region of interest. Subtracted cDNA probes enriched for sequences expressed from the nonoverlap region are isolated by the addition of streptavidin followed by organic extraction. These subtracted cDNA probes are used to screen a cDNA library prepared from an appropriate tissue, and candidate cDNAs are mapped by Southern hybridization to determine whether they are encoded within the nonoverlap region. This technique is an attractive alternative to other methods for identifying genes in a genomic region because it does not require that the gene of interest be localized to a minimal genetic interval, and the genomic DNA encompassing the region need not be cloned. Additionally, the method detects only expressed genes, simplifying the search for candidate genes within a genomic region.

Journal Article↗

PubMed1991

Hybrid Bacillus (1-3,1-4)-beta-glucanases: engineering thermostable enzymes by construction of hybrid genes.

Hybrid (1-3,1-4)-beta-glucanase genes were constructed by extension of overlapping segments of the (1-3,1-4)-beta-glucanase genes from Bacillus amyloliquefaciens and B. macerans generated by the polymerase chain reaction (PCR). Four hybrid genes were expressed in Escherichia coli cells. The mature hybrid enzymes contain a 16, 36, 78, or 152 amino acid N-terminal sequence derived from B. amyloliquefaciens (1-3,1-4)-beta-glucanase followed by a C-terminal segment derived from B. macerans (1-3,1-4)-beta-glucanase. Biochemical characterization of parental and hybrid enzymes shows a significant increase in thermostability of three of the hybrid enzymes when exposed to an acidic environment thus combining two important enzyme characteristics within the same molecule. At pH 4.1, 85%-95% of the initial activity was retained after 1 h at 65 degrees C in contrast to 5% and 0% for the parental enzymes from B. amyloliquefaciens and B. macerans. After 60 min incubation at 70 degrees C, pH 6.0, the parental enzymes retained 5% or less of the initial activity whilst one of the hybrids still exhibited 90% of the initial activity. Of the parental enzymes B. macerans (1-3,1-4)-beta-glucanase had the lower specific activity while the hybrid enzymes exhibited specific activities that were 1.5- to 3-fold higher. These experimental results demonstrate that exchange of homologous gene segments from different species may be a useful technique for obtaining new and improved versions of biologically active proteins.

Amino Acid Sequence↗

PubMed2000

The effect of hybrid layer thickness on bond strength: demineralized dentin zone of the hybrid layer.

OBJECTIVES: The purpose of this study was to evaluate the correlation between hybrid layer thickness and bond strength using specimens acid-conditioned for varying lengths of time. METHODS: The dentin surfaces of human premolars, sectioned to remove the enamel from the labial surface, were conditioned with 35.0% phosphoric acid of an adhesive resin system (Scotchbond Multi-Purpose; 3M) for 15 (as directed by the manufacturer), 60, 120, or 180 s (experimental acid-conditioning times). The bonded specimens were then sectioned perpendicular to the adhesive interface to measure the hybrid layer thickness by SEM. The specimens for the micro-tensile test were sectioned perpendicular to the adhesive interface and trimmed to an hourglass-shape. Then, the micro-tensile test was performed at a crosshead speed of 1.0 mm/min. The bond strengths and hybrid layer thickness were statistically compared with Student's t-test (p < 0.05). All fractured surfaces were also observed by SEM. RESULTS: Significant differences between the groups exposed to acid for 15 and 60 s, and those exposed for 120 and 180 s were observed in hybrid layer thickness and bond strength (p < 0.05). SEM observation of the fractured surfaces revealed that a demineralized dentin zone without resin impregnation remained within the hybrid layer. SIGNIFICANCE: A demineralized dentin zone was formed in the bond structures after prolonged acid-conditioning, resulting in low bond strength. The shrinkage of the hybrid layer due to desiccation during the SEM examination process provided evidence of the presence of the demineralized dentin zone within the hybrid layer.

Acid Etching, Dental↗