Chromosomes and chromatine structural arrangement of genetic information Chromatin (Walther Flemming 1882) = DNA + associated proteins euchromatin x heterochromatin heterochromatin facultative x constitutive Chromatin (DNA + associated proteins) Genetic information = DNA sequence (change = mutation) - protein-coding, regulatory, RNA-coding - Epigenetic information (less stable, depends on location) - transcriptional activity, access of interacting proteins transcriptionally active decondensed, accessible x x transcriptionally inactive compact, unaccessible Epigenetic modifications of chromatin - epigenetic information can be mitotically and meiotically herritable (e.g. some changes in gene activity) - no change in primary DNA sequence - modifications of chromatin components: • DNA methylation • histone posttranslational modifications mutually interconnected! • histone types euchromatin heterochromatin Nucleosome octamer of histones (small alcaline proteins): 2 x H2A, 2 x H2B, (2 x H3, 2 x H4) + 147 bp DNA Varying composition: - histone variants (isoforms): CenH3, H3.3, H2A.Z - posttranslational modifications of histone proteins Structure of 30nm fibre Solenoid or ZigZag? - still unclear solenoid Li and Reinberg 2011 Higher structural order of chromatine - hypothetically loops with actively transcribed genes - insufficiently understood • MARs (matrix attachment regions) alt. SARs (scaffold attachment reg.) – harboring regions surrounding coding sequences to nuclear protein matrix – AT rich, colocalize with „insulators“ (sequences that prevent spreading of heterochromatin) – distances between 3 - 100kb • LBARs (loop basement attachment regions) - organize chromosomes to huge loops (distances 20kb až 100kb) example of hypothetical arrangement Interconnections betweeen nucleosomes - linker sequence between: length = multiple of 10 bp (20 to 90 bp) - average (most frequent) length - differences among species, tissues, … (20 bp yeast, 30 bp Arabidopsis, 40 bp mammels) - internucleosomal fragmentation yields: 167 – 237 bp ( frequent length of repeats) Interconnections betweeen nucleosomes Direct interactions - N-ends of H4 interact with H2A.H2B bodies in parallel fiber - presence of H2A.Z variant probably prevents parallel interaction Linker histone H1 - alcaline both ends (amino and carboxy) interaction with both histones and DNA - stabilization of higher structures (30nm), phosphorylated during cell cycle - length of linker sequence: longer - require H1, more compact – heterochromatin shorter – H1 less important, more decondensed, active chromatin Nucleosome position • arrangement on DNA is not random (but is changable) - DNA sequence - DNA methylation - histone modification/types - DNA transcription • regulation / modulation of transcription - „unstable nucleosom region“ (earlier „nucleosom-free region“) in front of transcription start site (mainly constitutively expressed genes) – - unstable nucleosomes with H3.3 and H2A.Z histones - surrounded with stably situated nucleosomes with H2A.Z - nukleosomes help to define exons (central location even without transcription!) Histone code - covalent posttranslatinal modifications (PTM) - modifications mainly on N-ends (out of core) - high complexity - „epigenetic instruction“ to manage with DNA Some histone PTMs are mutually interconnected and have multiple functions e.g. H2A phosphorylation – injured DNA labelling, but also role in regulation of transcription and spiralization and in PCD Rossetto et al. 2012 Histone code Phosphorylation – predominantly short-time transient label, various functions Acethylation – predominantly „executive modification“ for weakening interaction with DNA - K-Ac – specific interaction of bromodomain proteins – signal role ( stabile), both repressive and activating (~ depends on position) - K-Me – specific interaction with chromodomain and TUDORlike domain proteins - key role in regulation of DNA methylation and chromatine activity - H3K9me2, H3K4me3, H3K27me3 Methylation Reproduction of nucleosomes after replication - histone tetramers (H3/H4) and dimers (H2A.H2B) not divided between sister strands! - one strand – „parental histons“ (Asf1) de novo deposition (CAF1, Asf1) - H2A.H2B incorporated even later (post replication) Chromosomes NOR: 18S- 5,8S- 26S rDNA Caryotype – number, types and sizes of chromosomes Classical caryotype (metaphase) Flow caryotype (FISH labelling) Doležel et al. 1999 Chromosome number and sizes Number: 2 - 600 Size: 2,4 Mb Genlisea 30 Mb Arabidopsis 800 Mb Triticum What are the consequences? - different linkage groups (various gene recombination) - limited hybrid fertility, … Chromosome number differs between species • Extreme chromosome numbers – Haplopappus gracilis: 2n = 4 – Sedum suaveolens: 2n = cca 640 • Luzula sp.: – 2n = 6 až 66 – holocentric chromosomes – Chromosom size differes up to 60x L. pilosa (Cullis, Plant genomics and proteomics, 2004) L. elegans B chromosomes in plants - non-pair chromosomes in some species (1500 species – maize) - usually no protein-coding genes - usually negatively affect fitness (fertility) - not present in all individuals in population - parazitic DNA (?) Chromosome number and genome size Telomeres DNA-protein structures serving to maintain stability of chromosomal ends Repetitive sequences synthetized by telomerase after replication (TTTAGGG)n in Arabidopsis Some plants have typical mammalian sequence: (TTAGGG)n Keeping telomere length Telomerase - RT with an RNA template repeat number depends on: - species - developmental stage - cell type - chromosome (within a cell) Centromeres - attachement of chromatids - defined by presence of histone CenH3 - CenH3 – kinetochore – spindel fibers Types:- point (125 bp, yeast Saccharomyces) - holocentric chromosomes (CenH3 along whole chr.) e.g. Luzula – allows fragmentation - classical – region of different length formed with heterochromatin (repeats, TE) = epigenetically defined (neocentromeres) - various strenth in hybrids Chromosomes: ((telocentric, acrocentric, metacentric,submetacentric)) Crossing of WT Arabidopsis with a line carrying modified (weaker) CenH3 issues in haploid progeny – inefficient segregation of chromosomes (elimination) Ravi and Chan 2010 Chromosomal territories Regions in nucleus occupied with certain chromosome (postmitotic decondensation 2 hours, 2.5 fold increse) Rabl’s arrangement of chromosomes in interphase nuclei centromeres and telomeres localized in oposite sites (chrom. size above 500 Mb) WHY? Experimental confirmation of chromosomal territories - specific labelling of chromosomes (FISH) - laser injury, detection of reparation Cremer and Cremer 2010 Sex chromosomes in plants - sex determination (single locus or more loci) Sexuality in various taxons of plants evolved independently and repeatedly (5 % species, in about 75 % plant families) - Marchantia, Gingo, Silene, Rumex, Hop, Poplar … Sex chromosomes in plants Morphological classification of sex chromosomes - homomorphic - heteromorphic - polymorphic – more than two types: e.g. Rumex acetosa: male XY1Y2, female XX Humulus lupulus var. cordiflorus: male X1X2Y1Y2, female X1X1X2X2 Evolution of sex chromosomes Formation of sex chromosome and single sex individuals – primary mutations causing male and female sterility in loci in strong genetic linkage (intermediarily usually gynodioecy) model: female (XX) – an allele (in locus A) necessary for development of male sex organs is non-functional in X-chromosome ancestor (recessive allele) male XY – an allele (in locus B, linked with locus A) is mutated to suppress formation of female sex organs (dominant allele), this allele is linked with functional allele in locus A Evolutionary young – homomorphic (recombination only partially limited) Degenerations (inversions, TE amplification, deletions) - heteromorphic Splitting or translocations can issue in polymorhic Polyploidy complicates formation of sex chromosomes
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