1. Chromosomes and chromatine structural arrangement of genetic information

2. Chromatin (Walther Flemming 1882) = DNA + associated proteins euchromatin x heterochromatin heterochromatin facultative x constitutive

3. 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 x transcriptionally inactive decondensed, accessible x compact, unaccessible

4. DNA methylation histone posttranslational modifications mutually interconnected! histone types 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: euchromatin heterochromatin

5. Varying composition: - histone variants (isoforms): CenH3, H3.3, H2A.Z - posttranslational modifications of histone proteins Nucleosome octamer of histones (small alcaline proteins): 2 x H2A, 2 x H2B, (2 x H3, 2 x H4) + 147 bp DNA

6. Structure of 30nm fibre Solenoid or ZigZag? - still unclear solenoid Li and Reinberg 2011

7. LBARs (loop basement attachment regions) - organize chromosomes to huge loops (distances 20kb až 100kb) 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 Higher structural order of chromatine - hypothetically loops with actively transcribed genes - insufficiently understood example of hypothetical arrangement

8. 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)

9. 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

10. 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!)

11. Histone code - covalent posttranslatinal modifications (PTM) - modifications mainly on N-ends (out of core) - high complexity - „epigenetic instruction“ to manage with DNA

12. 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

13. 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 Methylation – signal role (  stabile), both repressive and activating (~ depends on position) - K-Me – specific interaction with chromodomain and TUDOR- like domain proteins - key role in regulation of DNA methylation and chromatine activity - H3K9me2, H3K4me3, H3K27me3

14. 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)

15. Chromosomes NOR: 18S- 5,8S- 26S rDNA

16. Caryotype – number, types and sizes of chromosomes Flow caryotype (FISH labelling) Doležel et al. 1999 Classical caryotype (metaphase)

17. 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, …

18. 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 (Cullis, Plant genomics and proteomics, 2004) L. pilosa L. elegans

19. 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 (?)

20. Chromosome number and genome size

21. 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

22. Telomerase - RT with an RNA template repeat number depends on: - species - developmental stage - cell type - chromosome (within a cell) Keeping telomere length

23. - 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 Centromeres Chromosomes: ((telocentric, acrocentric, metacentric,submetacentric))

24. Crossing of WT Arabidopsis with a line carrying modified (weaker) CenH3 issues in haploid progeny – inefficient segregation of chromosomes (elimination) Ravi and Chan 2010

25. Rabl’s arrangement of chromosomes in interphase nuclei centromeres and telomeres localized in oposite sites (chrom. size above 500 Mb) WHY? Chromosomal territories Regions in nucleus occupied with certain chromosome (postmitotic decondensation 2 hours, 2.5 fold increse)

26. Cremer and Cremer 2010 Experimental confirmation of chromosomal territories - laser injury, detection of reparation - specific labelling of chromosomes (FISH)

27. Sex chromosomes in plants Sexuality in various taxons of plants evolved independently and repeatedly (5 % species, in about 75 % plant families) - Marchantia, Gingo, Silene, Rumex, Hop, Poplar … - sex determination (single locus or more loci)

28. Sex chromosomes in plants Morphological classification of sex chromosomes - homomorphic - heteromorphic - polymorphic – more than two types: e.g. Rumex acetosa: male XY 1 Y 2, female XX Humulus lupulus var. cordiflorus: male X 1 X 2 Y 1 Y 2, female X 1 X 1 X 2 X 2

29. 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