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At least 163 records · Page 9Linked to original sources

PubMed2003

Production of fertile somatic hybrid plants between Oriental hybrid lily and Liliumxformolongi.

Somatic hybridizations via electrofusion were performed in combinations of Oriental hybrid lilies (cvs. Acapulco and Shirotae) and Liliumxformolongi hort. (cv. Hakucho). Electrofusion-treated protoplasts divided only under nurse culture. The divided protoplasts grew into calli on the culture medium containing picloram, and the calli were then transferred to the hormone-free culture medium for induction of plant regeneration. The regenerants were transferred to a greenhouse, and were grown until the flower stage. In the fusion combinations of Acapulco + Hakucho and Shirotae + Hakucho, four regenerants apparently showed different morphological features compared with their parents. The results of molecular analyses by means of cleaved amplified polymorphic sequences markers and flow cytometry confirmed that these regenerants were somatic hybrid plants. Furthermore, we examined the stability of the morphological features of the hybrids in the next generations. This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion.

Cell Division↗

PubMed1995

Human chromosome-specific changes in a human-hamster hybrid cell line (AL) assessed by fluorescent in situ hybridization (FISH).

PURPOSE: To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. METHODS AND MATERIALS: Synchronized human-hamster hybrid cells containing human chromosome 11 were obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%) were irradiated with 137Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0, and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells collected 16-20 h later; chromosomal spreads were prepared, denatured, and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. RESULTS: Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatible with mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosome per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosome was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosome was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40-60%) incidence of incomplete exchanges between human and hamster chromosomes. CONCLUSIONS: Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1 (translocations) were readily scored and found to equate with the complementary asymmetrical exchanges (dicentrics). That is, nonlethal stable changes, which might be of concern in carcinogenic processes, complement lethal, unstable changes. Interstitial deletions that may contribute to the loss of antioncogenes as well as to lethality are also readily detected with enhanced levels detected at higher doses. The high level of induced terminal deletions and of incomplete dicentrics and translocations indicate a partial failure of interaction between lesions induced in human and hamster DNA, and suggest that such interspecies interactions lack the fidelity of intraspecies DNA lesion interactions. This suggests caution in the use of such model systems as indicators of human cell responsiveness.

Animals↗

PubMed1984

Chromosomal sites of integration of simian virus 40 DNA sequences mapped by in situ hybridization in two transformed hybrid cell lines.

Simian virus 40 DNA sequences, integrated on human chromosome 7 in two transformed human-mouse hybrid somatic cell lines, were mapped to a specific chromosomal locus by in situ hybridization. To detect the integrated viral DNA by in situ hybridization, we increased the sensitivity of the technique by using as a probe 125I-labeled simian virus 40 cRNA (2 X 10(9) to 3 X 10(9) dpm/micrograms) prepared by in vitro transcription of simian virus 40 DNA with high-specific-radioactivity [125I]CTP. Although the viral nucleic acid was shown by blot hybridization in the two cell lines to be integrated in different restriction fragments, it was shown by in situ hybridization in the two cell lines to map to the same position, q31, on the long arm of human chromosome 7. The viral DNA integration sites were localized with a precision of +/- 1 silver grain diameter, equivalent to about 6.2 X 10(7) nucleotide pairs in the human genome. The procedures we describe may be adapted for localization of any gene on diploid chromosomes that can be cloned in a recombinant DNA vector.

Animals↗

PubMed2003

Molecular cytogenetic analysis of wheat-barley hybrids using genomic in situ hybridization and barley microsatellite markers.

In the present investigation, genomic in situ hybridization (GISH) and barley microsatellite markers were used to analyse the genome constitution of wheat-barley hybrids from two backcross generations (BC1 and BC2). Two BC1 plants carried 3 and 6 barley chromosomes, respectively, according to GISH data. Additional chromosomal fragments were detected using microsatellites. Five BC2 plants possessed complete barley chromosomes or chromosome segments and six BC2 plants did not preserve barley genetic material. Molecular markers revealed segments of the barley genome with the size of one marker only, which probably resulted from recombination between wheat and barley chromosomes. The screening of backcrossed populations from intergeneric hybrids could be effectively conducted using both genomic in situ hybridization and molecular microsatellite markers. GISH images presented a general overview of the genome constitution of the hybrid plants, while microsatellite analysis revealed the genetic identity of the alien chromosomes and chromosomal segments introgressed. These methods were complementary and provided comprehensive information about the genomic constitution of the plants produced.

Chimera↗

PubMed2002

Physical mapping of genes in somatic cell radiation hybrids by comparative genomic hybridization to cDNA microarrays.

BACKGROUND: Somatic cell mutants can be informative in the analysis of a wide variety of cellular processes. The use of map-based positional cloning strategies in somatic cell hybrids to analyze genes responsible for recessive mutant phenotypes is often tedious, however, and remains a major obstacle in somatic cell genetics. To fulfill the need for more efficient gene mapping in somatic cell mutants, we have developed a new DNA microarray comparative genomic hybridization (array-CGH) method that can rapidly and efficiently map the physical location of genes complementing somatic cell mutants to a small candidate genomic region. Here we report experiments that establish the validity and efficacy of the methodology. RESULTS: CHO cells deficient for hypoxanthine:guanine phosphoribosyl transferase (HPRT) were fused with irradiated normal human fibroblasts and subjected to HAT selection. Cy5-labeled genomic DNA from the surviving hybrids containing the HPRT gene was mixed with Cy3-labeled genomic DNA from normal CHO cells and hybridized to a microarray containing 40,185 cDNAs, representing 29,399 genes (UniGene clusters). The DNA spots with the highest Cy5:Cy3 fluorescence ratios corresponded to a group of genes mapping within a 1 Mb interval centered near position 142.7 Mb on the X chromosome, the genomic location of HPRT. CONCLUSION: The results indicate that our physical mapping method based on radiation hybrids and array-CGH should significantly enhance the speed and efficiency of positional cloning in somatic cell genetics.

Animals↗

PubMed1975

Expression of differentiated functions in hepatoma cell hybrids: IX extinction and reexpression of liver-specific enzymes in rat hepatoma-Chinese hamster fibroblast hybrids.

Most of the hybrid clones derived from a cross of Chinese hamster fibroblasts (DON) with rat hepatoma cells (Faza 967) showed preferential loss of rat chromosomes. Two of the hybrid clones retained the rat chromosomes, and both showed extinction of 4 liver-specific enzymes: aldolase B, liver alcohol dehydrogenase, and the inducible enzymes tyrosine aminotransferase and alanine aminotransferase. Subcloning of 1 of these hybrids, which contained 2 sets of hepatoma chromosomes and 1 set of hamster chromosomes, permitted the isolation of some clones which reexpressed 1 or more of the liver-specific enzymes. Liver alcohol dehydrogenase was the most frequently reexpressed enzyme and aldolase B the least. Tyrosine aminotransferase inducibility was reexpressed independently of basal activity, and the enzyme produced by the reexpressing hybrid cells was precipitated by a specific antiserum. No correlation was detected between the presence or absence of the marker chromosomes (large metacentrics) of the hamster parent and the extinction and reexpression of the hepatic enzymes. The results reported confirm and extend to interspecific hybrids the observation of the stable and independent reexpression of tissue-specific enzymes.

Alanine Transaminase↗

PubMed1985

Detection of sickle-cell mutation by electrophoresis of partial RNA:DNA hybrids following solution hybridization.

We have developed a method in which partially single-stranded (ss) DNA molecules containing a defined region of duplex RNA:DNA are electrophoretically separated in agarose gels. The partial hybrids are formed by solution hybridization with a uniform length RNA probe complementary to part of the DNA sequence of interest. Following hybridization, the RNA/DNA mixture is fractionated by agarose gel electrophoresis at high temperature to minimize intrastrand base pairing which causes mobility heterogeneity. Not requiring the steps of DNA transfer from the gel to a solid support and subsequent probing, pre-electrophoretic hybridization allows the direct identification of single-copy fragments. Conditions for the detection of single-copy genes in human DNA digested with specific restriction endonucleases were developed and applied to the diagnosis of sickle-cell disease. This method should be applicable for the analysis of DNAs of high complexity where the presence of DNA polymorphisms and interspersed repeated DNA sequences often make impossible the creation of complete RNA:DNA hybrids.

Alleles↗

PubMed1996

Reduced formamide content and hybridization temperature results in increased non-radioactive mRNA in situ hybridization signals.

To define conditions for highly sensitive non-radioactive mRNA in situ hybridization on cryostat sections the influence of decreased formamide content and hybridization temperature was studied. The examination was performed on fibromatosis nodules of palmar fibromatosis visualizing the beta actin mRNA of myofibroblasts. The results show that a decrease in formamide content and hybridization temperature is able to enhance the sensitivity of mRNA detection applicating digoxigenin labelled DNA oligodeoxynucleotide. The best hybridization signal could be obtained under formamide-free conditions. In conclusion, a simplified sensitive formamide-free mRNA in situ hybridization protocol using oligonucleotide probes on human tissue cryostat sections is presented. The negative formamide effect is seen as a result of the chemical interaction of formamide with nucleic acid strands. An omission of formamide is suggested if the target as well as the probe are single stranded.

Actins↗

PubMed1971

Expression of differentiated functions in hepatoma cell hybrids. I. Tyrosine aminotransferase in hepatoma-fibroblast hybrids.

The inducible enzyme tyrosine aminotransferase (TAT) has been investigated in hybrids between rat hepatoma cells and mouse fibroblasts. In the latter the TAT baseline activity is low, and in the presence of steroids does not change. By contrast, the hepatoma cells have high TAT activity, and this activity increases by a factor of 4-6 in the presence of steroids. The hybrid cells, like the fibroblasts, have low TAT activity and are not inducible. Heat inactivation curves demonstrate that the hybrid cells contain both parental forms of TAT, and therefore contain the parental genes specifying the enzyme. The presence in the hybrids of detectable rat TAT and the total absence of its inducibility suggest that a second gene is involved in the regulation of TAT inducibility, and that this gene is not expressed in the hybrids.

Animals↗

PubMed1972

Formation of hybrids between coliphage lambda and Salmonella phage P22 with a Salmonella typhimurium hybrid sensitive to these phages.

An unusual Salmonella typhimurium hybrid with sensitivity to coliphage lambda and salmonella phage P22 has been recovered from matings between an Escherichia coli K-12 Hfr donor and an S. typhimurium recipient. The hybrid is an excellent host for achieving genetic recombination between lambda and P22. Two broad classes of hybrid phages were isolated. The lambda-P22 hybrid class, which has the protein coat of lambda, contains at least the c region of P22. The P22-lambda hybrid class has the protein coat of P22 and has inherited at least the c marker of lambda.

Animals↗

PubMed1972

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

The synthesis of serum albumin has been studied in hybrids between well-differentiated rat hepatoma cells, which synthesize serum albumin, and mouse fibroblasts (3T3) that do not synthesize albumin. By immunodiffusion techniques with noncrossreacting antisera, the production of both rat and mouse albumin by the hybrids has been examined. Karyologically identified hybrids were produced between 3T3 cells and cells of a 1s hepatoma (Fu5) clone, and of a 2s hepatoma (2s Fu5-5cl.lE) clone. Each of the 3T3 x Fu5 hybrids produces only rat albumin. Among five 3T3 x 2s Fu5-5cl.lE hybrid clones isolated, one produces both rat and mouse albumin, two produce only mouse albumin, and two do not produce rat or mouse albumin.

Animals↗

PubMed1977

Genetics of the connective tissue proteins: assignment of the gene for human type I procollagen to chromosome 17 by analysis of cell hybrids and microcell hybrids.

Somatic cell hybrids between mouse and human cell lines have been used to identify the specific chromosome that governs the synthesis of type I procollagen. Fourteen hybrid clones and subclones were derived independently from crosses between mouse parents [LM (thymidine kinase-negative) or A9 (hypoxanthine phosphoribosyltransferase-negative)] and human cells (human diploid lung fibroblasts WI-38 or diploid skin fibroblasts GM5, GM17, and GM9). The cultures were labeled with [(3)H]proline in modified Eagle's medium without serum. Radioactive procollagens were purified from the medium by the method of Church et al. [(1974) J. Mol. Biol. 86, 785-799]. DEAE-cellulose chromatography was used to separate collagen and type I and type III procollagen. Human type I procollagen was assayed by double immunodiffusion analysis with type I procollagen antibodies prepared by immunizing rabbits with purified human type I procollagen. These analyses combined with karyology and isozyme analyses of each hybrid line have produced evidence for the assignment of the gene for human type I procollagen to chromosome 17. A human microcell-mouse hybrid cell line containing only human chromosome 17 was positive for human type I procollagen, lending further support to the assignment of the human type I procollagen gene to chromosome 17. Finally, by using a hybrid line containing only the long arm of human chromosome 17 translocated onto a mouse chromosome, the type I procollagen gene can be assigned more specifically to the long arm of chromosome 17.

Animals↗

PubMed1996

Peptide nucleic acid pre-gel hybridization: an alternative to southern hybridization.

We have found that it is possible to use labeled peptide nucleic acid (PNA)-oligomers as probes in pre-gel hybridization experiments, as an alternative for Southern hybridization. In this technique, the PNA probe is hybridized to a denatured DNA sample at low ionic strength and the mixture is loaded directly on to an electrophoresis system for size separation. Ensuing gel electrophoresis separates the single-stranded DNA fragments by length. The neutral backbone of PNA allows for hybridization at low ionic strength and imparts very low mobility to excess PNA. Detection of the bound PNA is possible by direct fluorescence detection with capillary electrophoresis, or the DNA/PNA hybrids can be blotted onto a membrane and detected with standard chemiluminescent techniques. Efficient single bp discrimination was achieved routinely using both capillary and slab-gel electrophoresis.

Blotting, Southern↗

PubMed1981

Clonal analysis of F1 hybrid helper T cells. I-A subregion-encoded hybrid determinants restrict the activity of keyhole limpet hemocyanin-specific helper T cells.

Clonal expansion of isolated precursors to helper T cells was induced in limiting dilution cultures of keyhole limpet hemocyanin (KLH)-primed F1 hybrid lymph node cells. Progeny of each isolated precursor was tested for helper activity by transfer to independent cultures with hapten-primed B cells of either parental or F1 hybrid origin. The major histocompatibility complex (MHC) restriction specificity of each F1 hybrid helper T clone was determined. To assess the contribution of I-A and I-E subregion-encoded genes to the expression of these restriction elements, helper T cell cultures derived from F1 hybrids between strains with recombinant H-2 haplotypes were analyzed. Parental and unique F1 hybrid MHC determinants that are encoded entirely within the I-A subregion were found to restrict the activity of KLH-specific helper T cells.

Animals↗

PubMed1989

Detection of hepatitis delta virus in serum and liver tissue by molecular hybridization. Validation of a rapid spot-hybridization technique.

Serologic tests for detection of delta hepatitis virus (HDV) antigen and antibody have recently been supplemented with a Northern blot hybridization assay for HDV RNA. However, this technique is cumbersome for analysis of multiple samples. In order to simplify detection of HDV RNA, the authors have tested a spot-hybridization method with a new HDV cDNA probe. Their method has proved to be rapid, sensitive, and specific for HDV RNA even when less ultracentrifugation was used for recovering serum RNA. Results for HDV RNA were concordant by both spot and Northern blot hybridization in 12 serum samples from patients with known delta hepatitis, whereas in seven cases spot-hybridization was superior in detecting liver HDV RNA. The concordance between HDV RNA by spot hybridization and delta antigen was complete, whereas that between delta IgM (44% overall) or IgG (67% overall) was less strong. The authors' observations indicate that this new technology permits detection of HDV RNA with relative ease and could be applicable to the evaluation of large numbers of cases with delta hepatitis.

Animals↗

PubMed2006

Genome-wide patterns of expression in Drosophila pure species and hybrid males. II. Examination of multiple-species hybridizations, platforms, and life cycle stages.

Species often produce sterile hybrids early in their evolutionary divergence, and some evidence suggests that hybrid sterility may be associated with deviations or disruptions in gene expression. In support of this idea, many studies have shown that a high proportion of male-biased genes are underexpressed, compared with non-sex-biased genes, in sterile F1 male hybrids of Drosophila species. In this study, we examined and compared patterns of misexpression in sterile F1 male hybrids of Drosophila simulans and 2 of its sibling species, Drosophila mauritiana and Drosophila sechellia, at both the larval and adult life stages. We analyzed hybrids using both commercial Drosophila melanogaster microarrays and arrays we developed from reverse transcriptase-polymerase chain reactions of spermatogenesis and reproduction-related transcripts from these species (sperm array). Although the majority of misexpressed transcripts were underexpressed, a disproportionate number of the overexpressed transcripts were located on the X chromosome. We detected a high overlap in the genes misexpressed between the 2 species pairs, and our sperm array was better at detecting such misexpression than the D. melanogaster array, suggesting possible weaknesses in the use of an array designed from another species. We found only minimal misexpression in the larval samples with the sperm array, suggesting that disruptions in spermatogenesis occur after this life stage. Further study of these misexpressed loci may allow us to identify precisely where disruptions in the spermatogenesis pathway occur.

Animals↗

PubMed1999

Does interspecific hybridization influence evolutionary rates? An experimental study of laboratory adaptation in hybrids between Drosophila serrata and Drosophila birchii.

The low initial fitness of progeny from interspecific crosses in animals and the rarity of interspecific hybridization in natural environments have led to a debate about the evolutionary importance of this phenomenon. Here we directly assess the effects of hybridization between Drosophila serrata and Drosophila birchii on evolutionary rates. We looked at the effects on laboratory adaptation over 30 generations in two laboratory environments, one of which involved nutrition and temperature stress. Laboratory adaptation occurred over time in both environments as reflected by a marked change in viability. However, whilst hybrid lines at no stage performed poorly relative to parental lines, their rate of adaptation never exceeded that of the parentals. Thus, there was no evidence that hybridization increased evolutionary rates. Instead, hybrid lines converged phenotypically with one of the parental species.

Analysis of Variance↗

PubMed2003

Genetic dissection of hybrid incompatibilities between Drosophila simulans and D. mauritiana. III. Heterogeneous accumulation of hybrid incompatibilities, degree of dominance, and implications for Haldane's rule.

The genetic basis of Haldane's rule was investigated through estimating the accumulation of hybrid incompatibilities between Drosophila simulans and D. mauritiana by means of introgression. The accumulation of hybrid male sterility (HMS) is at least 10 times greater than that of hybrid female sterility (HFS) or hybrid lethality (HL). The degree of dominance for HMS and HL in a pure D. simulans background is estimated as 0.23-0.29 and 0.33-0.39, respectively; that for HL in an F1 background is unlikely to be very small. Evidence obtained here was used to test the Turelli-Orr model of Haldane's rule. Composite causes, especially, faster-male evolution and recessive hybrid incompatibilities, underlie Haldane's rule in heterogametic male taxa such as Drosophila (XY male and XX female). However, if faster-male evolution is driven by sexual selection, it contradicts Haldane's rule for sterility in heterogametic-female taxa such as Lepidoptera (ZW female and ZZ male). The hypothesis of a faster-heterogametic-sex evolution seems to fit the current data best. This hypothesis states that gametogenesis in the heterogametic sex, instead of in males per se, evolves much faster than in the homogametic sex, in part because of sex-ratio selection. This hypothesis not only explains Haldane's rule in a simple way, but also suggests that genomic conflicts play a major role in evolution and speciation.

Animals↗