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  • 1
    ISSN: 1432-2048
    Keywords: 5-Azacytidine ; DNA methylation ; DNA (tandem repeats) ; Nicotiana (tissue culture) ; Plant regeneration (tobacco)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previously, we established experimental conditions allowing us to induce hypomethylation of tandem arrays of highly repetitive DNA sequences in the Nicotiana tabacum L. nuclear genome (M. Bezděk et al. 1991, Planta 184, 487–490). In this paper, we demonstrate that loci containing the highly repetitive sequences of the HRS60 family can maintain the induced hypomethylated state through protoplast regeneration, non-differentiated callus growth, and plant regeneration. The hypomethylation, induced with 5-azacytidine and monitored on these sequences, did not substantially alter the capacity of calli to produce plants. It can be concluded that, in contradistinction of multiple copies of transgenes or ectopic genes which are usually recognized as methylation targets, endogenous tandem repeats, such as the HRS60, present at 105 copies in the genome, can escape de-novo methylation.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1432-2048
    Keywords: Key words: DNA methylation ; Facultative heterochromatin ; Gagea ; Endosperm nucleus (pentaploid)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract. Pentaploid endosperm nuclei in certain Gagea species exhibit large masses of sticky and dense chromatin, not observed in somatic nuclei. These heterochromatin masses most probably stem from the triploid chalasal polar nucleus of the embryo sac, thus representing an example of facultative heterochromatinisation in plants. In the present investigation, we studied the nuclei in Gagea lutea (L.) Ker-Gawl. endosperm tissue. The position of the heterochromatin in interphase nuclei was observed by confocal laser scanning microscopy (CLSM) and the DNA methylation status of the euchromatin and heterochromatin was analysed by immunolabelling with an antibody raised against 5-methylcytosine (anti-5-mC). In young endosperms, heterochromatin was relatively dispersed, occupying some peripheral and inner parts of the nuclei. In a later endosperm development, the nuclei became smaller and more pycnotic, and the heterochromatin masses were placed predominantly near the nuclear periphery. The distribution of anti-5-mC labelling on the heterochromatic regions was unequal: some parts appeared hypermethylated while other parts were, like the euchromatin, not labelled. During mitosis, the labelling intensity of all the chromosomes was approximately the same, thus indicating that there are no cytologically detectable methylation differences among the individual sets of chromosomes. However, differences in the anti-5-mC signal intensity along individual chromosomes were observed, resulting in banding patterns with highly positive bands apparently representing constitutive heterochromatic regions. From these results it is obvious that facultative heterochromatinisation, in contrast to constitutive heterochromatinisation, need not be strictly accompanied by a prominent DNA hypermethylation.
    Type of Medium: Electronic Resource
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  • 3
    ISSN: 1432-2048
    Keywords: 5-Azacytidine ; DNA methylation ; DNA repeat (tandem) ; Hypomethylation ; Nicotiana (hypomethylation of DNA) ; Tissue culture (DNA methylation)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The methylation status and 5-azacytidine-induced hypomethylation of CCGG sites within a family of tandemly organized, highly repeated DNA sequences of the Nicotiana tabacum L. nuclear genome (HRS60 family) were studied. As shown by in-situ hybridization experiments, the HRS60 family is clustered in a few regions of some tobacco chromosomes. The DNAs of leaf-derived calli, leaf-derived calli cultured on media with 5-azacytidine, and leaves were cleaved with restriction endonucleases differing in the sensitivity to the methylation of cytosine. After electrophoresis and Southern blotting they were hybridized with the HRS60 probe. We show that (i) CpG dinucleotides, and partially also CpCpG trinucleotides, of the HRS60 family are methylated in DNAs of the non-treated calli and leaves, and (ii) that these DNA repeats are sensitive to the action of a hypomethylating drug, 5-azacytidine.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Planta 152 (1981), S. 215-224 
    ISSN: 1432-2048
    Keywords: Bromodeoxyuridine tolerance ; Bromouracil excision ; Cytokinin autotrophy ; DNA synthesis ; Fluorodeoxyuridine effect ; Nicotiana
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in “normal” hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1573-6849
    Keywords: DNA methylation ; Melandrium album (syn. Silene latifolia) ; sex chromosomes ; subtelomeric heterochromatin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A number of X chromosome DNA sequences have been isolated from a dioecious plant, Melandrium album (syn. Silene latifolia),using chromosome microdissection followed by degenerate oligonucleotide-primed polymerase chain reaction (DOP–PCR) amplification. Six DNA clones were selected and further characterized by DNA/DNA hybridization techniques to check their copy numbers, sex-specific methylation patterns, species specificity and positions on chromosomes. These clones were moderately to highly repetitive (approximately 103–105 copies per haploid genome) and none of them gave a positive signal on Northern blots. One of the clones yielded a sex-specific methylation pattern: its abundant non-methylated CCGG island was found only in males. All the clones also hybridized to two closely related dioecious Melandrium species (M. rubrum and M. dicline). Nucleotide sequences of two X-derived clones showed a number of internal short direct repeats; one of them strikingly resembled a plant conservative telomere sequence (TTTAGGG).None of the clones hybridized to the X chromosome only, but all were localized at the telomeric heterochromatic regions (DAPI C-bands) of both arms of a vast majority of M. album chromosomes using the fluorescencein situ hybridization (FISH) technique. However, the non-homologousarm of the Y chromosome (contrary to the arm homologous to the X chromosome,possessing the pseudoautosomal region) showed neither a DAPI C-banding-stained heterochromatin nor a FISH signal with any of the DNA probes tested,thus indicating its evolutionary diversification.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    Protoplasma 211 (2000), S. 116-122 
    ISSN: 1615-6102
    Keywords: Lilium longiflorum ; DNA methylation ; Histone H4 acetylation ; Pollen ; Vegetative nucleus ; Generative nucleus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The first pollen mitosis results in generative and vegetative cells which are characterised by a striking difference in their chromatin structure. In this study, histone H4 acetylation and DNA methylation have been analysed during pollen development inLilium longiflorum. Indirect immunofluorescence procedures followed by epifluorescence and laser scanning microscopy enabled a relative quantification of H4 acetylation and DNA methylation in microspores, immature binucleate pollen, mature pollen, and pollen tubes. The results show that histone H4 of the vegetative nucleus, in spite of its decondensed chromatin structure, is strongly hypoacetylated at lysine positions 5 and 8 in comparison with both the original microspore nucleus and the generative-cell nucleus. These H4 terminal lysines in the vegetative nucleus are, however, progressively acetylated during the following pollen tube growth. The DNA methylation analysis inversely correlates with the histone acetylation data. The vegetative nucleus in mature pollen grains is heavily methylated, but a dramatic nonreplicative demethylation occurs during the pollen tube development. Changes neither in H4 acetylation nor in DNA methylation have been found during development of the generative nucleus. The results obtained indicate that the vegetative nucleus enters the quiescent state (accompanied by DNA hypermethylation and H4 underacetylation) during the maturation of pollen grain which enables pollen grains a long-term survival without external source of nutrients until they reach the stigma.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Springer
    Molecular genetics and genomics 239 (1993), S. 219-224 
    ISSN: 1617-4623
    Keywords: DNA methylation ; In situ nick-translation ; Lyon hypothesis ; Sex chromosomes ; Melandrium album
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Melandrium album, a dioecious plant species, has two heteromorphic sex chromosomes with the XY constitution typical for male and the XX for female plants. This plant represents an experimental model system of sex determination in which the Y chromosome plays a strongly dominant male role. We present data on the overall transcriptional activities of M. album sex chromosomes. DNA methylation patterns were analysed directly at the level of chromosomes using in situ nick-translation of fixed root mitotic chromosomes after nuclease digestion and in vivo labelling with S-adenosyl-l-[methyl-3H] methionine as donor of methyl groups. Both techniques revealed that the two X chromosomes of female plants had different levels of DNA methylation. Cell treatment with a DNA hypomethylating drug, 5-azacytidine, significantly influenced the labelling densities. These results imply that in female M. album plants, one of the two X chromosomes may be hypermethylated and inactive as described for mammalian cells (Lyon hypothesis). A similar analysis made on male cells displayed a similar relative levels of methylation in autosomes and sex chromosomes, thus indicating the transcriptional activity of both Y and X male chromosomes.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Chichester [u.a.] : Wiley-Blackwell
    Developmental Genetics 15 (1994), S. 214-230 
    ISSN: 0192-253X
    Keywords: Sex determination ; angiosperms ; genetics ; white campion ; sex chromosomes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Most flowering plant species are hermaphroditic, but a small number of species in most plant families are unisexual (i.e., an individ-ual will produce only male or female gametes). Because species with unisexual flowers have evolved repeatedly from hermaphroditic progenitors, the mechanisms controlling sex determination in flowering plants are extremely diverse. Sex is most strongly determined by genotype in all species but the mechanisms range from a single controlling locus to sex chromosomes bearing several linked locirequired for sex determination. Plant hormones also influence sex expression with variable effects from species to species. Here, we review the genetic control of sex determination from a number of plant species to illustrate the variety of extant mechanisms. We emphasize species that are now used as models to investigate the molecular biology of sex determination. We also present our own investigations of the structure of plant sex chromosomes of white campion (Silene latifolia - Melan-drium album). The cytogenetic basis of sex determination in white campion is similar to mammals in that it has a male-specific Y-chromosome that carries dominant male determining genes. If one copy of this chromosome is in the genome, the plant is male. Otherwise it is female. Like mammalian Y-chromosomes, the white campion Y-chromosome is rich in repetitive DNA. We isolated repetitive sequences from microdissected Y-chromosomes of white campion to study the distribution of homologous repeated sequences on the Y-chromosome and the other chromosomes. We found the Y to be especially rich in repetitive sequences that were generally dispersed over all the white campion chromosomes. Despite its repetitive character, the Y-chromosome is mainly euchromatic. This may be due to the relatively recent evolution of the white campion sex chromosomes compared to the sex chromosomes of animals. © 1994 Wiley-Liss, Inc.
    Additional Material: 8 Ill.
    Type of Medium: Electronic Resource
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