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Chromosomal basis of heredity RNDr. Z.Polívková Lecture No135 – Course:Cell structure.

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Presentation on theme: "Chromosomal basis of heredity RNDr. Z.Polívková Lecture No135 – Course:Cell structure."— Presentation transcript:

1 Chromosomal basis of heredity RNDr. Z.Polívková Lecture No135 – Course:Cell structure

2 History of chromosomal study : Sutton and Boveri – chromosomes are related to heredity Painter – chromosomal number = 48 = uncorrect Tjio and Levan – chromosome number in man = Lejeune et al.- 1 st chromosomal abnormality = trisomy 21 in patients with Down syndrome Jacobs and Strong - 47, XXY karyotype in males with Klinefelter syndrome Ford et al. - monosomy X in females with Turner syndrome Patau et al. – trisomy 13 in patients with Patau syndrome Edwards et al. – trisomy 18 in patients with Edwards syndrome 1966 – Steel, Breg – determination of chromosome constitution from amniotic fluid

3 Cytogenetics – study of chromosomes Clinical cytogenetics- study of chromosomal abnormalities

4 DNA Histones – basic proteins H1,H2A, H2B,H3,H4 Non histones proteins – neutral or slightly acidic The whole length DNA 2 m Human genome contains structural genes = protein coding genes = small fraction of the genome (1.5%) Ultrastructure of chromosomes

5 Nucleosome DNA double helix + histone core Histone core = octamere of two copies of H2A, H2B, H3, H4 DNA double helix is winded around the histone core, spacer segment of DNA between two nucleosomes is free or associated with H1 histone (appearance of beads on a string) = condensation to 1/10 of native DNA length String of nucleosomes is coiled into solenoid (6 nucleosomes in each turn) = fundamental unit of chromatin fiber Organization of chromatin in interphase

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8 Condensation of chromatin into chromosomes Solenoid is packed into loops attached to the nonhistone protein scaffold (Laemli loops) = chromosome in prophase – 1/3000 of native length Chromosome in metaphase = nonhistone protein scaffold with loops is coiled into spiral structure of chromatids Many steps of coiling = DNA is shortened to 1/10000 of its native length

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10 Human chromosomes – morphology Chrom. metacentric submetacentric acrocentric centromere p q chromatids telomere satellite sat. stalk (NOR) p = short arm q = long arm NOR = nucleolus organizer region (rRNA genes)

11 Centromeres Centromere (primary constriction) = DNA + histones (mostly α-satellite DNA = large numbers of short tandemly repeated sequences) Kinetochore = complex proteinaceous structure at centromere – mediates attachment of spindle microtubules and chromosome movement in metaphase and anaphase Centromere malfunction → nondisjunction (error in distribution of chromosomes during division)

12 Nucleolus organizer Nucleolus - located in nucleus – not bounded by membrane = site of transcription and processing of rRNAs, site of assembly of rRNA and proteins into two ribosomal subunits (subunits join to form cytoplasmic ribosomes) nucleoli disappear during mitosis, formed at telophase at specific sites of acrocentric chromosomes (satellite stalks of chromosomes Nos 13,14,15,21,22 = nucleolus organizer region (NOR) nucleoli - tendency to fuse together – satellite association NORs contain tandemly repetitive ribosomal RNA gene clusters variability in the length of this region (number of rRNA genes on each acrocentric is variable ( copies)

13 Telomeres tandemly repeated TTAGGG/CCCTAA sequences ( several thousand times ) protects chromosome ends from degradation and from fusions (telomeric DNA is packed to loops and asociated with proteins – i.e. protected from exonucleases that attact free ends of DNA ) essential role in pairing of homologs in meiosis association of telomeres with nuclear envelope

14 Replication of telomeres: enzyme telomerase (= ribonucleoprotein complex = reverse transcriptase – synthesizing DNA from RNA template) telomerase is abundant in embryonic and cancer cells, expresed highly in stem cells low, almost undetectable activity in somatic cells reduction of telomere length after each round of replication → cellular aging, or senescene (Hayflick limit – cell dies after certain number of cell division - due to the shortening of chromosomal telomeres to critical length) cancer cells are immortal (high level of telomerase activity)

15 FISH with telomeric probes Mouse telocentric chromosomes

16 Human chromosomes: 22 pairs of autosomes 1 pair of gonosomes (heterochromosomes) Karyotype: man 46, XY, woman 46, XX Chromatin – consist of: basic proteins (histones), DNA, nonhistone proteins, small amount RNA Euchromatin (active form of chromatine, transcribed) despiralized in interphase spiralized in mitosis contains structural genes

17 Heterochromatin  repetitive sequences (in constitutive-stable heterochromatin)  not transcribed into mRNA (=inactive)  partially folded in interphase  late replicating  tendency to form condensed clumps adjacent to nuclear membrane Constitutive (stable) – at centromeres of all chromosomes, blocks of heterochromatin on 1q, 9q,16q, Yq (Y chromatin), tandemly repeated sequences (satellite DNA) length variability of heterochromatic parts - origin by unequal crossing-over

18 Facultative (reversible) - structurally euchromatin, but behaves as heterochromatin (potentially transcribable sequences that are specifically inactivated = one of two X chromosomes in women = genetically inactive, late replicating (replication at the end of S phase) = X chromatin (sex chromatin = Barr body)

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20 Karyotype 46,XX – G bands

21 Karyotype 46,XY – G bands

22 Heterochromatin 1.Richness in satellite DNA (= tandemly repeated sequences) - in constitutive heterochromatin 2.Stability: constitutive heterochromatin is stable, facultative is reversible (inactive X is reactivated before meiosis) 3.Staining: constitutive heterochromatin is strongly stained by C-band technique C-banding = specific staining of heterochromatic parts (=strong denaturation of all euchromatic parts – pale, resistant heterochromatin is darkly stained) 4. Polymorphism : constitutive heterochromatin is polymorphic in size and localization (instability of satellite DNA) – without phenotypic effect

23 Properties of heterochromatin: 1. condensation (both constitutive and facultative) 2. late replicating (both constitutive and facultative, inactive X replicates at the end of S phase) 3. methylation (on cytosines) 4. histones in heterochromatin are hypoacetylated (hyperacetylated histones are in active chromatin)

24 Histone acetylation removes positive charge of histones – thus reducing the force of attraction with DNA = open chromatin (active) Deacetylation of histones restores positive charge leading to close attraction betwen histones and DNA (condensed chromatin structure – inactive – not accesible for transcription factors) Numerous transcription factors have either activity Histon Acetyl Transferase (HAT) – in activation of transcription or Histon De-Acetylases (HDAC)- in repression of transcription HDAC = multiprotein complex - contains methyl cytosin binding proteins (MeCP1, MeCP2) - selectively bind methylated DNA HDAC is targeted to methylated DNA (CpG) Other histone modifications: phosphorylation, methylation – chrom. condensation Histone modification, DNA methylation and chromosome condensation

25 5. histones in heterochromatin are methylated on lysine - methylation of histones creates binding site for heterochromatic protein HP1 – role in organisation of heterochromatin 6. Heterochromatin is transcriptionally inactive constitutive heterochromatin does not contain any genes facultative: genes are not usually transcribed 7. Heterochromatin does not participate in genetic recombination polymorphism of heterochromatic regions - difficulties in homologous pairing 8. Tendency to agregate during interphase agregation of short arms of acrocentrics – nucleolus organiser region=NOR 9. Role of nuclear RNA in formation of facultative heterochromatin – X inactivation (mRNA – product of gene XIST)

26 Function of heterochromatin: 1. Heterochromatin and euchromatin occupy different domains. heterochromatin is on periphery of nucleus attached to nuclear membrane Active chromatin – central position in nucleus, it allows maximal efficiency of replication and transcription 2. Centromeric heterochromatin - role in centromeric function – in cohesion of sister chromatids and normal disjunction of chromatids 3. Role in epigenetic regulation of gene expression during differentiation: probably certain active genes are transported into heterochromatic domain to become inactive

27 X-inactivation – Lyon´s hypothesis (Lyon 1961) only one X is active in somatic cells of all mammalian females, second one (or all others) is inactive (methylated) = condensed in interphase = stained as X-chromatin = Barr body (described by Barr and Bertram in 1949) inactivation begins in early embryonic development (probably at to cell stage) inactivation is random (according to parental origin of X) inactivation is stable in all daughter cells (descendant of any cell that underwent X-inactivation) woman = mosaic of cell with inactive paternal and maternal X chromosomes

28 inactivation is not complete – some genes on X chrom. escape inactivation in oogenesis both X are active – reactivation before meiosis structurally abnormal X - nonrandom inactivation - chromosomal abnormality (rearrangement) balanced (no material additional, no material missing) – preferentially normal X is inactive - chromosomal abnormality unbalanced (gain or loss of genetic material) – abnormal X is inactive Nonrandom inactivation in case of chromosomal abnormalities = consequence of selection

29 Barr body

30 X M X P XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP XM XPXM XP X inactivation

31 X-inactivation centre on Xq133, gene XIST X- inactivation controlled by XIST mRNA - expressed only on the inactive chromosome Screening method for sex determination - sex chromatin examination in some sports disciplines

32 Unbalanced aberration - terminal deletion Xp

33 Nonrandom X inactivation (detected by BUDr method): unbalanced aberration - terminal deletion of Xp The abnormal X chromosome is inactive - late replicating (pale) in all cells tested

34 Unbalanced aberration –ring chromosome X

35 Nonrandom X inactivation ( detected by BUDr method): unbalanced aberration – ring chromosome X The abnormal X chromosome is inactive - late replicating (pale) in all cells tested

36 Balanced aberration – reciprocal X/A translocation

37 Nonrandom X inactivation (detected by BUDr method): Balanced aberration – X/autosomal reciprocal translocation Normal X is inactive - late replicating (pale) in all cells tested

38 Thompson &Thompson: Genetics in medicine,7th ed. Chapter 2: The human genome and chromosomal basis of heredity, parts: Organization of human chromosomes, Human karyotype Chapter 6(part): X chromosome inactivation + informations from presentation


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