Chromosomal basis of heredity

RNDr. Z.Polívková

Lecture No135 – Course:Cell structure

History of chromosomal study:

1903 - Sutton and Boveri – chromosomes are related to heredity

1923 - Painter – chromosomal number = 48 = uncorrect

1956 - Tjio and Levan – chromosome number in man = 46

1959 - Lejeune et al.- 1st 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

1960 - 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

Cytogenetics – study of chromosomes

Clinical cytogenetics- study of chromosomal abnormalities

DNA Histones – basic proteins

H1,H2A, H2B,H3,H4 Non histones proteins – neutral or slightly acidic

The whole length DNA 2 mHuman genome contains 20-25 000 structural genes = protein coding genes = small fraction of the genome (1.5%)

Ultrastructure of chromosomes

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

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

Human chromosomes – morphologyChrom. metacentric submetacentric acrocentric

centromere

p

q chromatids

telomere

satellite

sat. stalk (NOR)

p = short armq = long armNOR = nucleolus organizer region (rRNA genes)

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)

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 (10 -100 copies)

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

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)

FISH with telomeric probes

Mouse telocentric chromosomes

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

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

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)

Karyotype 46,XX – G bands

Karyotype 46,XY – G bands

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

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)

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

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)

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

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 1000- to 2000- 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

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

Barr body

XM XP

XM XPXM XP

XM XPXM XPXM XP

XM XPXM XP XM XPXM XP

XM XP

XM XP

XM XP XM XP XM XP

X inactivation

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

Unbalanced aberration - terminal deletion Xp

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

Unbalanced aberration –ring chromosome X

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

Balanced aberration – reciprocal X/A translocation

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

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

+ informations from presentation

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