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  • 1
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Plant Physiology and Plant Molecular Biology 44 (1993), S. 53-76 
    ISSN: 1040-2519
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Plant, cell & environment 9 (1986), S. 0 
    ISSN: 1365-3040
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract Cationic potential-sensitive dyes have previously been used to selectively stain mitochondria in living animal cells (Johnson, Walsh & Chen, 1980; Johnson et al., 1981). The present work demonstrates that the cyanine dye 3,3′-dihexyloxacarbocyanine iodide (DiOC6(3)) can also be used as a mitochondrial stain in living plant cells. The stained mitochondria were easily visualized by fluorescence microscopy. The accumulation of DiOC6(3) in mitochondria seemed to be potential-dependent since it was prevented by protonophores, valinomycin and inhibitors of electron transport. It was often observed that DiOC6(3) also stained the nuclear membrane of some cells. This fluorescence, limited to the perinuclear region, was possibly due to a potential across one or both nuclear membranes, although it was not completely dissipated by any of the ionophores or inhibitors tested. Our observations demonstrate the usefulness of using DiOC6(3) for studying relative membrane potentials of plant mitochondria and, perhaps, other organelles and membrane systems in living plant cells.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    Plant, cell & environment 11 (1988), S. 0 
    ISSN: 1365-3040
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Abstract. To study whether an electrical potential difference exists across the nuclear envelope or inner nuclear membrane of plant cells, the authors have used an optical probe of membrane potential, the cationic fluorescent dye, DiOC6(3) (MW = 572.5). This dye was microinjected into the nucleoplasm of isolated Acetabularia nuclei (which are still surrounded by a thin layer of cytoplasm) and its subnuclear localization visualized by fluorescence microscopy. Striking differences, which seemed to be correlated with the developmental stage of the isolated nucleus, were observed. In nuclei isolated from cells at the stage of early cap stage formation, the dye was restricted to the nuclear envelope. In nuclei isolated from cells with intermediate or fully developed caps, there was increased nucleoplasmic staining, and the staining of the envelope was frequently diminished or abolished. In all nuclei, the dye remained within the nucleus after injection. Cytoplasmic staining was only observed when nuclei isolated from cells at the stage of early cap formation were incubated in a hyper- or hypo-tonic medium. Various ionophores, injected before the dye into the nucleoplasm, had no effect on the subsequent nuclear localization of DiOC6(3), although they did rapidly induce nucleolar condensation in nuclei isolated from cells at the stage of early cap formation. The results suggested that the electrical properties of Acetabularia nuclear envelopes or inner nuclear membranes change during cell maturation. Furthermore, the retention of the dye in the nucleoplasm under isotonic conditions indicated that the nuclear pores were not open channels for molecules of this size.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract We cloned and characterized a new highly repetitive, species-specific DNA sequence from turkey (Meleagris gallopavo). This repeat family, which accounts for approximately 5% of the turkey genome, consists of a 41 bp repeated element that is present in tandem arrays longer than 23 kb. In situ hybridization to turkey metaphase chromosomes (2n=80) demonstrated that this sequence was located primarily on certain microchromosomes: approximately one-third of the 66 microchromosomes showed a positive signal. With respect to the macrochromosomes, hybridization was seen only in a pericentric position on nos. 2 and 3. The turkey microchromosome (TM) sequence shares motifs (alternating A3–5 and T3–5 clusters separated by 6–8 bp) that have been found previously in other avian tandemly repeated elements, e.g. a chicken microchromosome sequence, and W (female) chromosome-specific sequences of chicken and turkey. However, the TM sequence does not cross-hybridize under moderately stringent conditions with these other sequence. The spread and amplification of related repetitive sequence elements on microchromosomes and W chromosomes is discussed.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. We have used combined fluorescent and genomic in situ hybridization (FISH/GISH) together with 4′,6-diamidino-2-phenylindole (DAPI) counterstaining to determine simultaneously the chromosome integration site and subgenomic allocation of a transgene in-sert in amphidiploid tobacco (Nicotiana tabacum, 2n = 4x = 48). The procedure provides sufficient information on physical markers to identify at least 20 out of 24 chromosome pairs of two tobacco cultivars commonly used in studies on transgene expression and silencing (cv. Petit Havana SR1 and cv. Gatersleben). The chromosomes can be distinguished on the basis of diploid parental ancestry, size, morphology, the presence of rDNA loci and/or intergenomic exchanges, and the DAPI banding pattern, which is shown here for the first time for N. tabacum. From a single ISH experiment, it should now be possible in most cases to identify a tobacco chromosome carrying a transgene insert, thus permitting systematic studies of how the chromosome location of transgenes influences expression levels.
    Type of Medium: Electronic Resource
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  • 6
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract To study whether specific DNA sequences are associated with nuclear membranes, residual DNA was extracted from DNase-treated nuclear envelopes prepared from erythrocytes of adult chickens (Gallus domesticus). This DNA was then blunt-end ligated into a bacterial plasmid vector. DNA blot analysis and nucleotide sequence determination revealed that approximately 30% of the cloned fragments consisted of different multiples of a 41–42 bp tandemly repeated, partially symmetrical sequence. In situ hybridization to chicken chromosomes demonstrated that the sequence was located primarily on microchromosomes, although some hybridization was also observed to macrochromosomes 7 and 8. Digestion of chicken DNA with any of a number of restriction enzymes did not completely reduce the intensity of a high molecular weight band to which the repeated sequence hybridized. These results, along with those obtained from in situ hybridization, suggested that many copies of this sequence are organized into large tandem arrays, and are not dispersed in many shorter repetitive blocks throughout the chicken genome. Although the repetitive sequence constituted approximately 10% of the chicken genome, it did not hybridize to quail or turkey DNA.
    Type of Medium: Electronic Resource
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  • 7
    ISSN: 1432-0886
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract An approach is described for the detection of a unique sequence, the T-DNA region of the Agrobacterium rhizogenes root-inducing (Ri) plasmid, in plant chromosomes by in situ hybridization. This sequence was introduced into the Crepis capillaris genome (2n=6) by infecting Crepis stem segments with A. rhizogenes. Roots growing from the infection site contain T-DNA and synthesize mannopine, which can be used as a convenient biochemical marker for T-DNA transformation. Southern analysis of DNA isolated from one transformed Crepis root line verified the presence of a single copy of T-DNA (approximate size 17 kb) per diploid Crepis genome. To localize T-DNA, both DNA and RNA probes, labelled with either tritium or biotin, were hybridized to Crepis chromosomes prepared from transformed root tips by a novel spreading method. Biotinylated probes were visualized using reflection-contrast microscopy. In the hybridization experiments described, T-DNA was detected in one homologue of chromosome 3, where it could be assigned to a paracentromeric position in the neighbourhood of the nucleolar organizing region. These results demonstrate that it is possible to localize unique sequences in plant chromosomes by in situ hybridization.
    Type of Medium: Electronic Resource
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  • 8
    ISSN: 1432-203X
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A genetic test was performed on seeds from 283 transgenic tobacco plants obtained by T-DNA transformation. Seeds from self-fertilized transgenic plants were germinated on kanamycin-containing medium, and the percentage of seeds which germinated, as well as the ratio of kanamycin-resistant to kanamycin-sensitive seedlings were scored. Nine categories of transformants could be distinguished according to the number of loci into which T-DNA had inserted, and according to the effects of T-DNA integration on seed or seedling development. In most of the plants, T-DNA was inserted into a single site; others contained multiple independent copies of T-DNA. The number of T-DNA integration sites was found to be independent of whether a binary vector system or a cointegrate Ti plasmid had been used to obtain the transgenic plant. Loss of marker genes or marker gene expression from generation to generation appeared to be a quite frequent event. Plants which appeared to be insertional recessive embryo-lethal mutants did not exhibit this trait in the next generation.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Springer
    Cellular and molecular life sciences 54 (1998), S. 94-103 
    ISSN: 1420-9071
    Keywords: Key words. Cosuppression; DNA methylation; epigenetics; gene silencing; isochores; paramutation; trans-inactivation; transposable elements.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Linked and unlinked copies of transgenes and related endogenous genes in plants can be epigenetically silenced by homology-based mechanisms that operate at either the transcriptional or post-transcriptional level. Transcriptional inactivation is associated with promoter homology and meiotically heritable methylation. Post-transcriptional silencing requires homology in protein-coding regions and is fully reversed during meiosis. Recently, the notion that both of these processes reflect the action of different host defence systems has been strengthened: (i) Obvious parallels have emerged between promoter homology-dependent silencing/methylation of transgenes and paramutation of endogenous genes that contain transposable elements in their promoters; (ii) remarkable similarities have been observed between post-transcriptional silencing involving transgenes and natural forms of virus resistance in nontransgenic plants. These results and others implicate two distinct cellular defence responses in transgene silencing. One is active in the nucleus and is manifested by transgene methylation, a reaction that might have originated as a means to oppose the spread of transposable elements. A second line of defence resides in the cytoplasm and operates through enhanced RNA turnover, a process that might help plants overcome viral infection.
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Springer
    Plant molecular biology 14 (1990), S. 323-332 
    ISSN: 1573-5028
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A zein gene (Z4) promoter containing 886 bp upstream from the transcription start site has been shown previously to be active specifically in the endosperm of transgenic tobacco seeds. To investigate the region required for this tissue-specific activity, deletions of the Z4 promoter were constructed and placed upstream of the β-glucuronidase (GUS) reporter gene. When these deletions were tested in transgenic tobacco plants, seed-specific GUS activity, which reached a peak between 15 and 19 DAP, was observed for promoters extending from −886 to −174. Interestingly, the 174 bp promoter lacked the complete 15 bp consensus sequence found in the same position in all zein genes so far sequenced. With the next shorter promoter in the deletion series (79 bp), which just included the CAAT and TATA elements, negligible GUS activity was observed in seeds. The results demonstrated that 174 bp upstream of the transcription start site are sufficient for tissue-specific and temporally regulated activity of the Z4 promoter in tobacco. At most, two-fold enhanced activity was observed with additional 5′ sequences up to −886.
    Type of Medium: Electronic Resource
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