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Results for “hybridization”

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PubMed1973

Studies of nondefective adenovirus 2-simian virus 40 hybrid viruses. VII. Characterization of the simian virus 40 RNA species induced by five nondefective hybrid viruses.

Five nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid viruses have been isolated and found to contain segments of SV40 DNA covalently linked to Ad2 DNA. The quantity of SV40 DNA present is a stable characteristic of each hybrid virus, and varies from less than 5% (in Ad2(+)ND(3)) to more than 30% (in Ad2(+)ND(4)) of the SV40 genome. We have characterized the SV40 portions of these hybrids by relating the SV40-specific RNA sequences transcribed in cells infected with each hybrid virus to those transcribed in cells infected with each of the other hybrid viruses and with SV40 itself. RNA-DNA hybridization-competition experiments indicate that the number of unique SV40 RNA sequences transcribed in infected cells is proportional to the size of the SV40 DNA segment contained within each hybrid and, in the case of the three hybrids which induce detectable SV40-specific antigens, to the number of SV40 antigens induced. Furthermore, the SV40-specific RNA sequences transcribed from any one of the hybrids are completely represented in the RNA transcribed from all other hybrids with longer SV40 segments. Thus, the SV40 DNA regions in the five hybrid viruses appear to contain some nucleotide sequences in common. The SV40-specific RNA transcribed from Ad2(+)ND(4), the hybrid containing the largest SV40 segment, is qualitatively similar to the SV40-specific RNA transcribed early (i.e., prior to viral DNA replication) in SV40 lytic infection. Thus, it appears that no significant amount of late SV40 DNA is transcribed during infection by any of the five nondefective Ad2-SV40 hybrid viruses.

Adenoviridae↗

PubMedDate not supplied

Hybrids between rubella virus and a latent virus of baby hamster kidney cell line BHK21: characterization of rubella virus and type 2 hybrid virus genomes and determination of their physical homology.

The biochemical nature of rubella virus and type 2 hybrid virus, which is a recombinant between rubella virus and a latent retrovirus of BHK21 cells, has been characterized. Type 2 hybrid virus carries DNA polymerase able to copy exogenous DNA. However, disrupted type 2 hybrid virions do not synthesize detectable amounts of DNA using the endogenous viral RNA or synthetic poly(rA)/oligo(dT) primed as a template. Thus, the type 2 hybrid virus DNA polymerase has no detectable reverse transcriptase activity. Rubella virus and type 2 hybrid virus RNA can serve as templates for avian myeloblastosis virus (AMV) reverse transcriptase, although they are inefficient. The addition of oligo(dT) to these viral RNA showed no significant stimulation of their template activity for AMV reverse transcriptase. The oligo(dT)-cellulose affinity column bound neither rubella virus nor type 2 hybrid virus RNA. This suggests that both RNA genomes have a very short poly(A) tail at their 3' end. Thus, complementary DNA (cDNA) synthesis by AMV reverse transcriptase using oligo(dT) primers showed no preferential reverse transcription from the genomic 3' terminus and produced only short cDNA fragments (about 200 nucleotides). We cross-hybridized these short cDNA fragments with their viral RNA, assuming that they are copies of random sites of the genome. These cDNA-RNA hybridization analyses of physical homology between type 2 hybrid virus and rubella virus genomes revealed that about 70% of the type 2 hybrid virus genome is derived from about an 85% portion of the rubella virus genome. These values indicate that the size of the type 2 hybrid virus genome is about 21% larger than that of the rubella virus genome. Co-sedimentation studies of these viral RNA by sucrose density gradient centrifugation confirmed that the molecular weight of type 2 hybrid virus RNA is 20% higher than that of rubella virus RNA. We propose a genomic structure of the type 2 hybrid virus taking into account both physical and biochemical data.

Animals↗

PubMed2004

Hybrid zone structure and the potential role of selection in hybridizing populations of native westslope cutthroat trout (Oncorhynchus clarki lewisi) and introduced rainbow trout (O. mykiss).

Introgressive hybridization is a common feature of many zones of contact between divergent lineages of fishes. This is particularly common when taxa that are normally allopatric come into artificial (human-induced) secondary contact. We examined 18 native populations of westslope cutthroat trout (Oncorhynchus clarki lewisi, WCT) to determine the extent of introgressive hybridization with introduced rainbow trout (O. mykiss, RBT) and the genetic structure of hybridizing populations in the upper Kootenay River, southeastern British Columbia, Canada. Using four diagnostic nuclear loci we calculated a hybrid index, inbreeding coefficient, FIS, and the linkage disquilibrium correlation coefficient, Rij, for each locality to determine the distribution of genotypes in each population. We also categorized the 142 hybrid individuals found across localities into four hybrid classes based on their genotypes. The majority of localities (11/18) showed a unimodal distribution of genotypes skewed towards genotypes of WCT. Two localities, however (lower Gold Creek and Lodgepole Creek) showed a flat to bimodal distribution and one site (lower Bull River) showed a unimodal distribution skewed towards RBT genotypes. The majority of hybrid individuals were classified genotypically as WCT backcrosses (59%) and post-F1 individuals (24%). We found a skewed ratio of pure WCT to pure RBT (17:1) and only four F1 hybrids (3%), suggesting that the spread of RBT alleles may be facilitated by hybrids straying to neighbouring populations. We also tested for the action of selection in one population using cohort analyses, but found little evidence of differential selection between pure WCT and hybrid individuals. Pooled across age classes there were significant differences in genotypic frequencies among loci suggesting differential introgression. There was no asymmetry to the hybridization between rainbow trout and westslope cutthroat trout because both species' mitochondrial DNA haplotypes were observed at similar frequencies in the hybrids. Our analyses suggest that hybrid swarms are likely to form in the upper Kootenay River drainage and that certain native WCT populations in British Columbia are at risk of local genomic extinction.

Age Factors↗

PubMed1995

A hybrid-hybrid matrix method for 3D NOE-NOE data analysis.

A hybrid-hybrid matrix method is described that quantitatively analyzes 3D NOE-NOE NMR data. Experimental 3D data are merged with simulated 3D data to create a hybrid 3D NOE-NOE spectrum. This is then deconvoluted into a 2D hybrid NOESY spectrum. The deconvoluted, 2D hybrid NOESY spectrum can then be merged with other 2D NOESY experimental data along with additional simulated 2D data as necessary to create a hybrid-hybrid 2D NOE volume matrix. This hybrid-hybrid volume matrix is then used with the complete relaxation program, MORASS, to calculate a rate matrix, and the resulting distances taken from the off-diagonal cross-relaxation rates can then be utilized in a distance geometry or restrained molecular-dynamics refinement of the structure. This process is repeated until a satisfactory agreement between the calculated and observed 3D volumes is obtained. This hybrid-hybrid matrix method retains computational efficiency and utilizes the resolution of the 3D data set while retaining any information content of the available 2D data. The initial tests of the deconvolution algorithm give high correlation results even with the introduction of random error into the 3D data set. Our results suggest that the hybrid-hybrid matrix method for analysis of 3D NOE-NOE spectra may provide a viable tool in the refinement of large molecules.

Algorithms↗

PubMed1991

Intergeneric somatic hybridization in Gramineae: somatic hybrid plants between tall fescue (Festuca arundinacea Schreb.) and Italian ryegrass (Lolium multiflorum Lam.).

Tall fescue (Festuca arundinacea Schreb.) protoplasts, inactivated by iodoacetamide, and non-morphogenic Italian ryegrass (Lolium multiflorum Lam.) protoplasts, both derived from suspension cultures, were electrofused and putative somatic hybrid plants were recovered. Two different genotypic fusion combinations were carried out and several green plants were regenerated in one of them. With respect to plant habitus, leaf and inflorescence morphology, the regenerants had phenotypes intermediate between those of the parents. Southern hybridization analysis using a rice ribosomal DNA probe revealed that the regenerants contained both tall fescue- and Italian ryegrass-specific-DNA fragments. A cloned Italian ryegrass-specific interspersed DNA probe hybridized to total genomic DNA from Italian ryegrass and from the green regenerated somatic hybrid plants but not to tall fescue. Chromosome counts and zymograms of leaf esterases suggested nuclear genome instability of the somatic hybrid plants analyzed. Four mitochondrial probes and one chloroplast DNA probe were used in Southern hybridization experiments to analyze the organellar composition of the somatic hybrids obtained. The somatic hybrid plants analyzed showed tall fescue, additive or novel mtDNA patterns when hybridized with different mitochondrial gene-specific probes, while corresponding analysis using a chloroplast gene-specific probe revealed in all cases the tall fescue hybridization profile. Independently regenerated F. arundinacea (+) L. multiflorum somatic hybrid plants were successfully transferred to soil and grown to maturity, representing the first flowering intergeneric somatic hybrids recovered in Gramineae.

Cells, Cultured↗

PubMed2005

Ecophysiology of first and second generation hybrids in a natural plant hybrid zone.

Hybrids between related species vary widely in relative fitness, and that fitness can depend upon the environment. We investigated aspects of physiology that might influence fitness patterns in a plant hybrid zone. Seeds of Ipomopsis aggregata, I. tenuituba, F1 hybrids, F2 hybrids, and offspring of crosses between natural hybrids were planted into the relatively mesic site of origin for I. aggregata and the drier site for natural hybrids. We measured rates of photosynthesis (Amax), transpiration (E), instantaneous (A/E) and long-term (delta13C) indices of water use efficiency (WUE), and leaf nitrogen and carbon. We also examined correlations of these traits with plant size. Photosynthetic rate and A/E were higher in vegetative than flowering plants. WUE varied between sites and years, but differences among genotypic classes were spatially and temporally consistent. Instantaneous WUE was higher for F1 hybrids than for the average of the parental species, thereby showing heterosis. There was no evidence of hybrid breakdown, as WUE was no different in the F2 than the average across the F1 and parental species. Nor did WUE depend on cross direction in producing F1 progeny. Carbon isotope discrimination revealed higher long-term water use efficiency in I. tenuituba than I. aggregata. Leaf nitrogen was higher in I. tenuituba than I. aggregata, and higher in offspring of natural hybrids than in the F2. Results indicate heterosis for water use efficiency, with no hybrid breakdown. Heterosis in WUE may help to explain the relatively high survival of both reciprocal F1 hybrids in dry sites within the natural hybrid zone.

Analysis of Variance↗

PubMed1999

Homoeologous chromosome pairing in the distant hybrid Alstroemeria aurea x A. inodora and the genome composition of its backcross derivatives determined by fluorescence in situ hybridization with species-specific probes.

A distant hybrid between two diploid species (2n = 2x = 16), Alstroemeria aurea and A. inodora, was investigated for homoeologous chromosome pairing, crossability with A. inodora and chromosome transmission to its BC1 offspring. Fluorescence in situ hybridization (FISH) with two species-specific probes, A001-I (A. aurea specific) and D32-13 (A. inodora specific), was used to analyse chromosome pairing in the hybrid and the genome constitution of its BC1 progeny plants. High frequencies of associated chromosomes were observed in both genotypes of the F1 hybrid, A1P2-2 and A1P4. In the former, both univalents and bivalents were found at metaphase I, whereas the latter plant also showed tri- and quadrivalents. Based on the hybridization sites of DNA probes on the chromosomes of both parental species, it was established that hybrid A1P4 contains a reciprocal translocation between the short arm of chromosome 1 and the long arm of chromosome 8 of A. inodora. Despite regular homoeologous chromosome pairing in 30% of the pollen mother cells, both hybrids were highly sterile. They were backcrossed reciprocally with one of the parental species, A. inodora. Two days after pollination, embryo rescue was applied and, eventually, six BC1 progeny plants were obtained. Among these, two were aneuploids (2n = 2x + 1 = 17) and four were triploids (2n = 3x = 24). The aneuploid plants had originated when the interspecific hybrid was used as a female parent, indicating that n eggs were functional in the hybrid. In addition, 2n gametes were also functional in the hybrid, resulting in the four triploid BC1 plants. Of these four plants, three had received 2n pollen grains from the hybrid and one a 2n egg. Using FISH, homoeologous crossing over between the chromosomes of the two parental species in the hybrid was clearly detected in all BC1 plants. The relevance of these results for the process of introgression and the origin of n and 2n gametes are discussed.

Brazil↗

PubMed1986

Tumorigenicity of T lymphoma/T lymphoma hybrids and T lymphoma/normal cell hybrids.

Stable hybrids formed between clones of established murine T-cell lymphoma lines, and between lymphoma clones and normal spleen or thymus cells were examined for their tumorigenic properties by intravenous (i.v.) and intradermal (i.d.) inoculation into syngeneic AKR mice. Fusion parents consisted of T lymphoma clones of high and low tumorigenicity derived from the SL 12 cell line. In addition, normal spleen cells and thymocytes were fused with poorly tumorigenic T-lymphoma clones. Hybrids tested by i.v. inoculation of 10(6) cells to syngeneic hosts showed that fusion between the lymphoma cells resulted in hybrids which displayed the phenotype of the highly tumorigenic parent. Also, it was shown that fusion of poorly tumorigenic lymphoma cells with normal spleen cells resulted in hybrids with enhanced tumorigenicity. Fusion of poorly tumorigenic lymphoma cells with normal thymocytes resulted in hybrids with the highest tumorigenic potential. The pattern of spread for the tumor/tumor hybrid was that of the highly tumorigenic parent. Tumor spread patterns for the spleen/tumor hybrids were different from those of the thymocyte/tumor hybrids. Intradermal inoculation of 10(5) cells from tumor/spleen or tumor/thymocyte hybrids revealed differences in latent periods between parental and hybrid cells, the tumor/thymocyte hybrids having the shortest latent period. Surface marker studies and T-cell antigen receptor mRNA determinations in the tumor cell/normal cell hybrids indicated that the normal parent was a cell of immature phenotype. Therefore, high tumorigenicity is a dominant characteristic, and poorly tumorigenic but "immortal" T lymphoma cells can derive characteristics which increase their in vivo growth capacity from the putative immature normal cells with which they selectively fuse.

Animals↗

PubMed1993

Hybrid lethal systems in the Drosophila melanogaster species complex. II. The Zygotic hybrid rescue (Zhr) gene of D. melanogaster.

Hybrid females from Drosophila simulans females x Drosophila melanogaster males die as embryos while hybrid males from the reciprocal cross die as larvae. We have recovered a mutation in melanogaster that rescues the former hybrid females. It was located on the X chromosome at a position close to the centromere, and it was a zygotically acting gene, in contrast with mhr (maternal hybrid rescue) in simulans that rescues the same hybrids maternally. We named it Zhr (Zygotic hybrid rescue). The gene also rescues hybrid females from embryonic lethals in crosses of Drosophila mauritiana females x D. melanogaster males and of Drosophila sechellia females x D. melanogaster males. Independence of the hybrid embryonic lethality and the hybrid larval lethality suggested in a companion study was confirmed by employing two rescue genes, Zhr and Hmr (Hybrid male rescue), in doubly lethal hybrids. A model is proposed to explain the genetic mechanisms of hybrid lethalities as well as the evolutionary pathways.

Animals↗

PubMed2001

Complex epistasis and the genetic basis of hybrid sterility in the Drosophila pseudoobscura Bogota-USA hybridization.

We analyzed the genetic basis of postzygotic isolation between the Bogota and USA subspecies of Drosophila pseudoobscura. These subspecies diverged very recently (perhaps as recently as 155,000 to 230,000 years ago) and are partially reproductively isolated: Bogota and USA show very little prezygotic isolation but form sterile F1 males in one direction of the hybridization. We dissected the basis of this hybrid sterility and reached four main conclusions. First, postzygotic isolation appears to involve a modest number of genes: we found large chromosome regions that have no effect on hybrid fertility. Second, although apparently few in number, the factors causing hybrid sterility show a remarkably complex pattern of epistatic interaction. Hybrids suffer no hybrid sterility until they carry the "right" allele (Bogota vs. USA) at at least four loci. We describe the complete pattern of interactions between all chromosome regions known to affect hybrid fertility. Third, hybrid sterility is caused mainly by X-autosomal incompatibilities. Fourth, hybrid sterility does not involve a maternal effect, despite earlier claims to the contrary. In general, our results suggest that fewer genes are required for the appearance of hybrid sterility than implied by previous studies of older pairs of Drosophila species. Indeed, a maximum likelihood analysis suggests that roughly 15 hybrid male steriles separate the Bogota and USA subspecies. Only a subset of these would act in F1 hybrids.

Animals↗

PubMed2005

Recent, allopatric, homoploid hybrid speciation: the origin of Senecio squalidus (Asteraceae) in the British Isles from a hybrid zone on Mount Etna, Sicily.

Homoploid hybrid speciation occurs through stabilization of a hybrid segregate (or segregates) isolated by premating and/or postmating barriers from parent taxa. Theory predicts that ecological and spatial isolation are of critical importance during homoploid hybrid speciation, and all confirmed homoploid hybrid species are ecologically isolated from their parents. Until recently, such species have been identified long after they originated, and consequently it has not been possible to determine the relative importance of spatial and ecological isolation during their origin. Here we present evidence for the recent origin (within the past 300 years) of a new homoploid hybrid species, Senecio squalidus (Asteraceae), in the British Isles, following long-distance dispersal of hybrid material from a hybrid zone between S. aethnensis and S. chrysanthemifolius on Mount Etna, Sicily, Italy. Historical records show that such hybrid material from Sicily was introduced to the Oxford Botanic Garden in Britain in the early part of the 18th century and that S. squalidus began to spread from there after approximately 90 years. A survey of randomly amplified polymorphic DNA/intersimple sequence repeats (RAPD/ISSR) marker variation demonstrated that S. squalidus is a diploid hybrid derivative of S. aethnensis and S. chrysanthemifolius that grow at high and low altitudes, respectively, on Mount Etna and that form a hybrid zone at intermediate altitudes. Senecio squalidus contained 11 of 13 RAPD/ISSR markers that were recorded at high frequency in S. chrysanthemifolius but were absent or occurred at low frequency in S. aethnensis, and 10 of 13 markers for which the reverse was true. Bayesian admixture analysis showed that all individuals of S. squalidus surveyed were of mixed ancestry with relatively high mean proportions of ancestry derived from both S. chrysanthemifolius and S. aethnensis (0.644 and 0.356, respectively). We argue that long-distance isolation of hybrid material from its parents on Mount Etna would have helped favor the origin and establishment of S. squalidus in the British Isles, regardless of whether the initial hybrid material introduced to Britain was preadapted to local conditions.

Altitude↗

PubMed1987

Inhibition of hybrid resistance by 5-fluorouracil. Destruction of a Thy-1+ Lyt-1+2- spleen cell implicated in the expression of hybrid resistance.

Hybrid resistance designates the poor proliferation of parent-strain bone marrow cells injected into lethally irradiated hybrids. While testing a variety of oncostatic drugs for their capacity to inhibit hybrid resistance, we found that 5-fluorouracil (5-FU) injected into B6D2F1 hybrids three days before grafting enhances the growth of parent-strain B6 bone marrow cells. Hybrid resistance can be restored in these 5-FU-treated B6D2F1 recipients by the injection of spleen cells from untreated (normal) B6D2F1 mice. Treatment of these normal B6D2F1 spleen cells with anti-Thy-1 or anti-Lyt-1 antibodies plus complement abolishes their capacity to restore resistance, whereas treatment with anti Lyt-2 antibody plus complement has no effect. It is known that the rejection of parent-strain bone marrow cells by the F1 hybrid is a multistep process in which more than one cell type is involved, and that the final effector cell is asialo-GMI+, cyclophosphamide-sensitive, and responsive to IFN (possibly the natural killer cell). The Thy-1+Lyt-1+2- spleen cell we describe here, which is eliminated by the 5-FU, doesn't seem to be the final effector cell for hybrid resistance since transfer of spleen cells from cyclophosphamide-treated (final-effector-cell-depleted) B6D2F1 hybrids into 5-FU treated B6D2F1 mice restores hybrid resistance just as well as do normal B6D2F1 spleen cells; and, when injected into 5-FU-treated B6D2F1 hybrids, the IFN inducer pI:pC also restores resistance. The cell we now describe would be a third cell type, probably responsible for the initial recognition of grafted parent-strain bone marrow cells, which triggers the mechanism of hybrid resistance. This hypothesis could explain the observed specificity of parent-strain bone marrow graft rejection by F1 hybrids.

Animals↗

PubMed2005

Effects of grazing residues or feeding corn from a corn rootworm-protected hybrid (MON 863) compared with reference hybrids on animal performance and carcass characteristics.

One grazing and two feeding experiments were conducted to compare the feeding value of corn residue or corn grain from a genetically enhanced corn hybrid (corn rootworm-protected; event MON 863) with nontransgenic, commercially available, reference hybrids. In Exp. 1, two 13.7-ha fields, containing corn residues from either a genetically enhanced corn root-worm-protected hybrid (MON 863), or a near-isogenic, nontransgenic control hybrid (CON) were divided into four equal-sized paddocks. Sixty-four steer calves (262 +/- 15 kg) were stratified by BW and assigned randomly to paddock to achieve a stocking rate of 0.43 ha/steer for 60 d, with eight steers per paddock and 32 steers per hybrid. A protein supplement was fed at 0.45 kg/steer daily (DM basis) to ensure protein intake did not limit performance. Steer ADG did not differ (P = 0.30) between steers grazing the MON 863 (0.39 kg/d) and CON (0.34 kg/d) corn residues for 60 d. The four treatments for the feeding experiments (Exp. 2 and 3) included two separate reference hybrids, the near-isogenic control hybrid (CON), and the genetically enhanced hybrid (MON 863) resulting in two preplanned comparisons of CON vs. MON 863, and MON 863 vs. the average of the reference hybrids (REF). In Exp. 2, 200 crossbred yearling steers (365 +/- 19 kg) were fed in 20 pens, with five pens per corn hybrid. In Exp. 3, 196 crossbred yearling steers (457 +/- 33 kg) were fed in 28 pens, with seven pens per corn hybrid. In Exp. 2, DMI and G:F did not differ (P > 0.10) between MON 863 and CON; however, steers fed MON 863 had a greater (P = 0.04) ADG than steers fed CON. Gain efficiency was greater (P = 0.05) for MON 863 cattle than for REF cattle in Exp. 2, but other performance measurements (DMI and ADG) did not differ (P > 0.10) between MON 863 and REF. No differences (P > 0.10) were observed for performance (DMI, ADG, and G:F) between MON 863 and CON or MON 863 and REF in Exp. 3. In terms of carcass characteristics, no differences (P > 0.10) were observed between MON 863 and CON, as well as MON 863 and REF, for marbling score, LM area, or 12th rib fat thickness in both Exp. 2 and 3. Overall, performance was not negatively affected in the corn residue grazing or feedlot experiments, suggesting the corn rootworm-protected hybrid (event MON 863) is similar to conventional, nontransgenic corn grain and residues when utilized by beef cattle.

Animal Feed↗

PubMed2000

Morphological and molecular evidence for hybridization and introgression in a willow (Salix) hybrid zone.

Hybrid zones provide biologists with the opportunity to examine genetic and ecological interactions between differentiated populations. Accurate identification of hybrid genealogies is considered a necessary prerequisite to understanding observed patterns of hybridization-related phenomena. We analysed molecular and morphological data from individuals in a hybrid zone between two species of willows (Salix sericea Marshall and S. eriocephala Michaux) and report the use of randomly amplified polymorphic DNA (RAPD), chloroplast DNA (cpDNA), and ribosomal DNA (rDNA) markers, as well as vegetative morphology and foliar chemistry data to identify individuals in terms of hybrid genealogy and to infer the direction and extent of backcrossing and introgression within the hybrid zone. A novel version of a maximum likelihood estimate approach (developed for this study) was used to calculate hybrid index scores from RAPD marker data; this method produced results similar to those obtained using traditional arithmetic methods. Distribution of rDNA, cpDNA, and chemistry data were examined within the graphical context of RAPD-based hybrid index score histograms and principal component analyses (PCA) on RAPD and morphology data. Seven of the 21 plants classified as S. eriocephala in the field were possible introgressants. Another plant presented an unequivocal example of backcrossed S. sericea chemistry and RAPD markers. Inter- and intraspecific chloroplast diversity found within the hybrid zone suggests both historic introgression (perhaps in a glacial refugium), and contemporary hybridization. Patterns of inheritance and expression within the hybrid zone suggest that morphological characters are often not expressed in a simple additive fashion, and problems associated with both morphological and molecular data are considered.

Crosses, Genetic↗

PubMed1990

Comparison of Southern transfer hybridization and dot filter hybridization for detection of cervical human papillomavirus infection with types 6, 11, 16, 18, 31, 33, and 35.

A commercial dot filter hybridization kit (Virapap Kit) was compared with Southern transfer hybridization for the detection of seven types of human papillomavirus (HPV) in cervical specimens from 450 consecutive females attending a sexually transmitted diseases clinic. In comparison with Southern transfer hybridization, performed with the same probes used in the dot filter kit, the sensitivity, specificity, and positive and negative predictive values of dot filter hybridization were 90%, 94%, 74%, and 98%, respectively. Among patients with cervical cytologic dysplasia, HPV DNA was detected in 44% by dot filter hybridization and in 35% by Southern transfer hybridization. Although 26% of specimens positive by dot filter hybridization were not confirmed by Southern transfer hybridization, cervical dysplasia was detected in 5 (25%) of 20 with HPV DNA detected by dot filter hybridization alone, compared with 25 (8%) of those with no definitive evidence of HPV by either method (P = 0.009) and with 16 (30%) of 53 with HPV DNA detected by both methods (P = 0.7). The kappa statistic for interobserver and intraobserver reproducibility for interpretation of blots was similar for the two methods. The dot filter hybridization method evaluated appears to be a satisfactory alternative to Southern transfer hybridization for detection of HPV DNA.

Adolescent↗

PubMed2006

Efficiency of model-based Bayesian methods for detecting hybrid individuals under different hybridization scenarios and with different numbers of loci.

Accurate detection of offspring resulting from hybridization between individuals of distinct populations has a range of applications in conservation and population genetics. We assessed the hybrid identification efficiency of two methods (implemented in the STRUCTURE and NEWHYBRIDS programs) which are tailored to identifying hybrid individuals but use different approaches. Simulated first- and second-generation hybrids were used to assess the performance of these two methods in detecting recent hybridization under scenarios with different levels of genetic divergence and varying numbers of loci. Despite the different approaches of the methods, the hybrid detection efficiency was generally similar and neither of the two methods outperformed the other in all scenarios assessed. Interestingly, hybrid detection efficiency was only minimally affected by whether reference population allele frequency information was included or not. In terms of genotyping effort, efficient detection of F1 hybrid individuals requires the use of 12 or 24 loci with pairwise F(ST) between hybridizing parental populations of 0.21 or 0.12, respectively. While achievable, these locus numbers are nevertheless higher than the number of loci currently commonly applied in population genetic studies. The method of STRUCTURE seemed to be less sensitive to the proportion of hybrids included in the sample, while NEWHYBRIDS seemed to perform slightly better when individuals from both backcross and F1 hybrid classes were present in the sample. However, separating backcrosses from purebred parental individuals requires a considerable genotyping effort (at least 48 loci), even when divergence between parental populations is high.

Animals↗

PubMed1996

A simple, reliable, and sensitive method for nonradioactive in situ hybridization: use of microwave heating to improve hybridization efficiency and preserve tissue morphology.

The digestion of fixed tissue sections is a critical step in the optimization of any in situ hybridization protocol. We describe a novel application of microwave oven heating to optimize mRNA detection in paraformaldehyde-fixed tissues by in situ hybridization using digoxigenin-labeled probes. This technique replaces protease digestion of fixed tissue sections with 10 min of microwave pretreatment, followed by either conventional hybridization or hybridization involving microwave incubation. This new technique has several advantages over the standard protease treatment-based methods presently in use. (a) Microwave oven heating is a simple, rapid, and highly reproducible technique. (b) Microwave pretreatment significantly increased the hybridization signal and reduced the background compared to conventional protease digestion. Consequently, the hybridization time required to obtain optimal mRNA detection was reduced to 30 min. (c) Ten minutes of microwave pretreatment produced an optimal hybridization signal in six different tissues using a variety of probes, demonstrating the general applicability of this technique. (d) Microwave heating of the probe during the hybridization step itself further reduced the hybridization time and substantially enhanced the hybridization signal obtained from proteinase K-digested tissue. (e) Microwave pretreatment caused no discernible loss of fine cell structure and tissue morphology compared to untreated tissue sections. In conclusion, microwave oven heating can replace the complicated strategies and poor reproducibility of protease treatment of tissue sections, resulting in a simple, rapid, more reliable and sensitive method that has general applicability for in situ hybridization.

Animals↗

PubMed2000

The hybrid-cluster protein ('prismane protein') from Escherichia coli. Characterization of the hybrid-cluster protein, redox properties of the [2Fe-2S] and [4Fe-2S-2O] clusters and identification of an associated NADH oxidoreductase containing FAD and [2Fe-2S].

Hybrid-cluster proteins ('prismane proteins') have previously been isolated and characterized from strictly anaerobic sulfate-reducing bacteria. These proteins contain two types of Fe/S clusters unique in biological systems: a [4Fe-4S] cubane cluster with spin-admixed S = 3/2 ground-state paramagnetism and a novel type of hybrid [4Fe-2S-2O] cluster, which can attain four redox states. Genomic sequencing reveals that genes encoding putative hybrid-cluster proteins are present in a range of bacterial and archaeal species. In this paper we describe the isolation and spectroscopic characterization of the hybrid-cluster protein from Escherichia coli. EPR spectroscopy shows the presence of a hybrid cluster in the E. coli protein with characteristics similar to those in the proteins of anaerobic sulfate reducers. EPR spectra of the reduced E. coli hybrid-cluster protein, however, give evidence for the presence of a [2Fe-2S] cluster instead of a [4Fe-4S] cluster. The hcp gene encoding the hybrid-cluster protein in E. coli and other facultative anaerobes occurs, in contrast with hcp genes in obligate anaerobic bacteria and archaea, in a small operon with a gene encoding a putative NADH oxidoreductase. This NADH oxidoreductase was also isolated and shown to contain FAD and a [2Fe-2S] cluster as cofactors. It catalysed the reduction of the hybrid-cluster protein with NADH as an electron donor. Midpoint potentials (25 degrees C, pH 7.5) for the Fe/S clusters in both proteins indicate that electrons derived from the oxidation of NADH (Em NADH/NAD+ couple: -320 mV) are transferred along the [2Fe-2S] cluster of the NADH oxidoreductase (Em = -220 mV) and the [2Fe-2S] cluster of the hybrid-cluster protein (Em = -35 mV) to the hybrid cluster (Em = -50, +85 and +365 mV for the three redox transitions). The physiological function of the hybrid-cluster protein has not yet been elucidated. The protein is only detected in the facultative anaerobes E. coli and Morganella morganii after cultivation under anaerobic conditions in the presence of nitrate or nitrite, suggesting a role in nitrate-and/or nitrite respiration.

Amino Acid Sequence↗