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Research paper thumbnail of Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in molecular biology, 2021

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

PLoS ONE, 2014

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are ... more Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-29-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/ nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.

Research paper thumbnail of Molekularna organizacija, epigenetički status i transkripcijska aktivnost 18S-5.8S-26S rDNA lokusa vrste Quercus robur

Položaj rDNA lokusa u jezgri, organizacija rDNA kromatina i prisutnost specificnih epigenetickih ... more Položaj rDNA lokusa u jezgri, organizacija rDNA kromatina i prisutnost specificnih epigenetickih oznaka pokazatelji su transkripcijske aktivnosti ili utisanosti rRNA gena. Istraživanjima provedenim u ovom doktorskom radu utvrđeno je da je u vrsti Q. robur, koja sadrži dva 18S-5.8S-26S rDNA lokusa, u normalnim fizioloskim uvjetima vjerojatno transkripcijski aktivan samo lokus NOR-1 koji pokazuje razlicite stupnjeve dekondenzacije kromatina, nalazi se uz jezgricu, boji se srebrom i hipometiliran je. Lokus NOR-2 je pak uvijek jako kondenziran, nalazi se udaljen od jezgrice cesto uz jezgrinu membranu, ne boji se srebrom, hipermetiliran je i obilježen epigenetickim modifikacijama karakteristicnim za heterokromatin sto ukazuje na njegovu transkripcijsku utisanost. Tretman stanica korjencica epigenetickim inhibitorom 5- aza-2'-dC dovodi do hipometilacije rDNA kromatina NOR-2 lokusa i njegovog pomicanja bliže jezgrici, ali ne i do dekondenzacija rDNA kromatina. U procesu utisavanja rDNA...

Research paper thumbnail of Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions

Genome biology and evolution, 2018

Epigenetic variation in natural populations with contrasting habitats might be an important eleme... more Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φS...

Research paper thumbnail of Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in molecular biology, 2021

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Nucleotide sequence, structural organization and length heterogeneity of ribosomal DNA intergenic spacer in Quercus petraea (Matt.) Liebl. and Q. robur L

Molecular genetics and …, 2009

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic ... more 18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcripti...

Research paper thumbnail of Molecular Cytogenetics (FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in Molecular Biology, 2014

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Molecular Cytogenetics (FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fl uorescence in situ hybridization (FI... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fl uorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

PLoS ONE, 2014

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are ... more Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-29-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/ nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.

Research paper thumbnail of Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in molecular biology, 2021

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

PLoS ONE, 2014

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are ... more Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-29-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/ nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.

Research paper thumbnail of Molekularna organizacija, epigenetički status i transkripcijska aktivnost 18S-5.8S-26S rDNA lokusa vrste Quercus robur

Položaj rDNA lokusa u jezgri, organizacija rDNA kromatina i prisutnost specificnih epigenetickih ... more Položaj rDNA lokusa u jezgri, organizacija rDNA kromatina i prisutnost specificnih epigenetickih oznaka pokazatelji su transkripcijske aktivnosti ili utisanosti rRNA gena. Istraživanjima provedenim u ovom doktorskom radu utvrđeno je da je u vrsti Q. robur, koja sadrži dva 18S-5.8S-26S rDNA lokusa, u normalnim fizioloskim uvjetima vjerojatno transkripcijski aktivan samo lokus NOR-1 koji pokazuje razlicite stupnjeve dekondenzacije kromatina, nalazi se uz jezgricu, boji se srebrom i hipometiliran je. Lokus NOR-2 je pak uvijek jako kondenziran, nalazi se udaljen od jezgrice cesto uz jezgrinu membranu, ne boji se srebrom, hipermetiliran je i obilježen epigenetickim modifikacijama karakteristicnim za heterokromatin sto ukazuje na njegovu transkripcijsku utisanost. Tretman stanica korjencica epigenetickim inhibitorom 5- aza-2'-dC dovodi do hipometilacije rDNA kromatina NOR-2 lokusa i njegovog pomicanja bliže jezgrici, ali ne i do dekondenzacija rDNA kromatina. U procesu utisavanja rDNA...

Research paper thumbnail of Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions

Genome biology and evolution, 2018

Epigenetic variation in natural populations with contrasting habitats might be an important eleme... more Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φS...

Research paper thumbnail of Molecular Cytogenetics (Fluorescence In Situ Hybridization - FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in molecular biology, 2021

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Nucleotide sequence, structural organization and length heterogeneity of ribosomal DNA intergenic spacer in Quercus petraea (Matt.) Liebl. and Q. robur L

Molecular genetics and …, 2009

18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic ... more 18S-5.8S-26S rDNA family comprises tandemly arranged, repeating units separated by an intergenic spacer (IGS) that contains transcription initiation/termination signals and usually repeating elements. In this study, we performed for the first time thorough sequence analysis of rDNA IGS region in two dominant European oaks, Quercus petraea and Q. robur, in order to investigate (1) if IGS sequence composition allows discrimination between these two species, and (2) if there is an rDNA length heterogeneity arising from IGS sequence. Two spacer length variants (slvs), 2 and 4 kb in length, were found in the genomes of both species. Inter-comparison of both slvs revealed no species-specificity in sequence or structural organization. Both slvs could be divided into four subregions; (1) the subrepeat region containing three repeated elements, (2) the AT-rich region containing matrix attachment sites and putative origin of replication, (3) the promoter region containing putative transcripti...

Research paper thumbnail of Molecular Cytogenetics (FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Methods in Molecular Biology, 2014

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FIS... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fluorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Molecular Cytogenetics (FISH and Fluorochrome Banding): Resolving Species Relationships and Genome Organization

Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fl uorescence in situ hybridization (FI... more Fluorochrome banding (chromomycin, Hoechst, and DAPI) and fl uorescence in situ hybridization (FISH) are excellent molecular cytogenetic tools providing various possibilities in the study of chromosomal evolution and genome organization. The constitutive heterochromatin and rRNA genes are the most widely used FISH markers. The rDNA is organized into two distinct gene families (18S-5.8S-26S and 5S) whose number and location vary within the complex of closely related species. Therefore, they are widely used as chromosomal landmarks to provide valuable evidence concerning genome evolution at chromosomal levels.

Research paper thumbnail of Inhibition of DNA Methylation Alters Chromatin Organization, Nuclear Positioning and Activity of 45S rDNA Loci in Cycling Cells of Q. robur

PLoS ONE, 2014

Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are ... more Around 2200 copies of genes encoding ribosomal RNA (rRNA) in pedunculate oak, Quercus robur, are organized into two rDNA loci, the major (NOR-1) and the minor (NOR-2) locus. We present the first cytogenetic evidence indicating that the NOR-1 represents the active nucleolar organizer responsible for rRNA synthesis, while the NOR-2 probably stays transcriptionally silent and does not participate in the formation of the nucleolus in Q. robur, which is a situation resembling the well-known phenomenon of nucleolar dominance. rDNA chromatin topology analyses in cycling root tip cells by light and electron microscopy revealed the minor locus to be highly condensed and located away from the nucleolus, while the major locus was consistently associated with the nucleolus and often exhibited different levels of condensation. In addition, silver precipitation was confined exclusively to the NOR-1 locus. Also, NOR-2 was highly methylated at cytosines and rDNA chromatin was marked with histone modifications characteristic for repressive state. After treatment of the root cells with the methylation inhibitor 5-aza-29-deoxycytidine, we observed an increase in the total level of rRNA transcripts and a decrease in DNA methylation level at the NOR-2 locus. Also, NOR-2 sites relocalized with respect to the nuclear periphery/ nucleolus, however, the relocation did not affect the contribution of this locus to nucleolar formation, nor did it affect rDNA chromatin decondensation, strongly suggesting that NOR-2 has lost the function of rRNA synthesis and nucleolar organization.