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MOG3 > Chromatin Arrangement > Flashcards

Chromatin Arrangement Flashcards

1
Q

Telomere?

A

Region of repetitive dna at ends of chromosomes
Highly stable - do not fuse with telomeres of other chromosomes
Protect important sequences at ends of chromosome from degradation

Maintain integrity of chromosome

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2
Q

Centromere?

A

Constricted region (or kinetochore) where spindle fibres attach

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3
Q

Sister chromatids?

A

Identical copies formed by dna replication - copies joined at centromere

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4
Q

Chromosome arms?

A

At each side of centromere
P (short) arm found at top
q (long) arm found at bottom

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5
Q

Human karyotype

A

22 pairs of autosomes
1 pair of sex chromosomes

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6
Q

I’m what order are chromosomes numbered?

A

Largest to smallest
Though in humans 21 is smaller than 22

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7
Q

Centromere position?

A

Metacentric:
P and q arm almost same size

Submetacentric:
Somewhat towards top - slightly smaller p than q arm

Acrocentric:
Centromere very close to one end
Very short p arm

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8
Q

Nuclear organisation centre?

A

NORs
Secondary constriction site on chromosome that act as nucleolar organisation sites

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9
Q

Giemsa staining?

A

Metaphase chromosome treated with trypsin to denature associated proteins
Stained with giemsa stain

Dark bands - represent heterochromatin - more condensed chromatin
(Gene poor, AT rich)

Lighter bands - represent euchromatin - less Condensed chromatin
(Gene rich, GC rich - more transcriptionally active)

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10
Q

FISH?

A

Fluorescent in situ hybridisation
Gene of interest cloned
Dna probe usually labelled with fluorescence
Probe and chromosomes from target cell are denatured to give ssDNA
Probe can hybridise to the homologous chromosome region
Shows where target gene is located on chromosome

Position of gene can be related to specific band of chromosome with giemsa band staining

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11
Q

Chromosome abnormality types?

A

Numerical:
Polyploidy
Aneuploidy (monosomy, trisomy)

Structural:
Deletions
Duplications
Inversions
Translocations

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12
Q

Polyploidy?

A

One or more additional chromosome sets e.g 3n triploids instead of 2n diploid

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13
Q

Aneuploidy?

A

Monosomy -
Loss of one chromosome from a homologous pair
Due to non- disjunction (failure of separating in meiosis)
Homologous chromosomes fail to separate - stay together in one of the daughters

Gives 2x gametes with trisomy (n+1)
And 2x with monosomy (n-1)

Can also happen in second division
Sister Chromatids fail to separate and remain in same daughter cell
Leads to 1 monosomy, 1 trisomy and 2 normal gametes

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14
Q

Aneuploidy disorders?

A

Sex chromosomes
Turner syndrome - manly female X0
Kleinfelter syndrome - mainly male XXY
Trisomy X - phenotypically normal, fertile, XXX or XYY

Autosomes:

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15
Q

Chromosome rearrangements?

A

Duplication
Deletion - duplication and deletion add/remove generic material - creates unbalance genome
Inversion - chromosome fragment reversed - genome can remain balanced - better tolerated than above - can disrupt genes though so can still cause issues
Translocation - chromosome fragment exchanged between two chromosomes

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16
Q

Classes of inversions?

A

Paracentric - occurs within one arm and preserves centromere location

Pericentric - happens across centromere - changes gene order and centromere location - and so will affect meiosis pairing and so can have effect on next generation

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17
Q

Mechanisms for chromosome rearrangement?

A
  1. DNA Breakage
    Deletion/duplication Need ds breaks (increased frequency in presence of mutagens)
  2. Non allelic homologous recombination (NAHR)
    Can happen between non homologous chromosomes with sequence similarity in regions
    Long copy repeats in the genome are misaligned if on non allelic chromosomes
    If gene is flanked by these they can recombine non allelically with another can delete (0 copies on one allele decreased gene dosage) or duplicate (extra copy of gene on allele - increased dosage) gene
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18
Q

Reciprocal translocation?

A

During prophase 1 of meiosis
Translocation happens between chromosome 1 and 2

Homology now present between them so two homologous pairs form a quadrivalent translocation heterozygote (all 4 together instead of just two in the bivalent)

Adjacent-1 segregation
2 homologous chromosomes segregated across the centromere
Leads to pair of 1T (translocated) and 2N (normal) pair and 1N and 2T
Unbalanced as deletion of one and extra chromosome on the other
Often unviable

Adjecant 2 segregation
Not through the centromeres
1T and 1N pair and 2T and 2N pair
Often unviable too (unbalanced)

Can alternatively segregate to give 1T and 2T pair and 1N and 2N pair
This is balanced
Chromosomes exchange parts with no loss or duplication

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19
Q

Robertsonian translocation?

A

DNA breaks
Might cause fusion between q arm of 21 and q arm of 14 forming a long metacentric chromosome
Other product is fused p arms giving a very small chromosome

21 and 14 are acrocentric so most genes in long q arms
So most genes are conserved in the long fused q arm chromosome and the mostly gene poor p arm fusion is lost after a few divisions

Balanced due to retaining of genetic info
But will causes issues with segregation in meiosis
E.g. extra chromosome 21 in zygote Causing downs syndrome

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20
Q

Philadelphia chromosome?

A

Exchange of part of 9q with 22q
Fused two genes
ABL from 9q
BCR from 22q
Fusion protein constitutively expressed
Causes immortality of leukaemia cells

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21
Q

How do new karyotypes evolve?

A

From fixed meitoic rearrangements
Needs to confer an advantage (rare)
Takes a long time to halpen

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22
Q

Synteny?

A

Conserved order of genes between species
E.g. blocks of chromosomes sharing gene order in mice and humans

23
Q

C-value (constant value) paradox?

A

Genome size does not correlate with complexity
Gene number does not significantly increase with genome size either

24
Q

C0t renaturation?

A

Denatured dna given time to cool done and renature to dsDNA
C0t value calculated by multiplying conc of DNA and time by the buffer factor
C0 x t x buffer factor

Renaturation curve
First bump on slope represents denaturing sequences that have more than one copy (repetitive sequences, reanneal qicker)
Lower bump shows abundant unique non repeating sequences

25
Q

What proportion of human genome is coding sequences?

A

1.5%
Rest if unique sequences:
RNA that we don’t know the function of (dark rna)
26% is non coding introns
Rest is repetitive sequences and transposable elements

26
Q

Structure of average gene?

A

25kb original mRNA transcript:
Introns
Non coding 5’ and 3’ UTRs
About 1.3kb left of protein coding exons

Outside of transcribed region
Enhancers
Promoters GC rich and contain CpG islands

Genes concentrated in areas of high GC content (euchromatin)
Unevenly dispersed around genome

27
Q

Examples of repetitive DNA

A

Gene families (not exactly repetitive - different genes related by sequence that arise from duplications)

Interspersed repeats

Tandem repeats

28
Q

Fates of duplicated genes?

A

Aquires loss of function mutation
Inactivated - Pseudogenization
Gene invisible now to natural selection
Can remain and acquire new mutations over time
Evolve by random mutation drift
Function of original gene retained by natural selection as is necessary to organism

Gene evolves new function
Neofunctionalisation
Alters activity and makes new protein
End up with new gene (in family?)

Function is divided between the two genes
Subfunctionalisation
Genes become dependent on each other
Both need to be activated and have complementary action

29
Q

Gene family benefit?

A

Provide variant proteins or large gene copy numbers

E.g. haemoglobin
Genes for alpha subunits located in cluster with beta subunit genes located in another

Beta subunits take over after gamma subunits stop being expressed after fetus is born

30
Q

Histones family?

A

Need lots of histones to organise genome - need to express lots of hisotne protein
Achieved by duplicated genes

31
Q

Interspersed repeats?

A

Non adjacent repeats
Dispersed throughout genome
SINEs -short, interspersed within and between genes (mainly between introns) - most are spread by retrotransposon and use rna intermediate to spread
LINEs - autonomous - contain everything they need to transpose including reverse transcriptase

32
Q

Variable number tandem repeats?

A

VNTRs
Interspersed thorough genome
Shirt sequences found multiple times in tandem
Variable number of repeats in tandem

3 groups by size
Satellite repeated 100,000s times - reach megabases to make centromer
AT Rich

Minisatellite 15-100 repeats (40-50bp per repeat)

Microsatellite <15 bp (2-3 repeated base pairs)

33
Q

How do VNTRs change in size?

A

Variable in number of repeats because:
Misalignment in meiosis
Unequal crossing over between two sequences
After recombination results in either expansion of tandem repeat or deletion in the other allele

DNA replication slippage:

Machinery falls off dna
Comes back on at wrong place due to repetivity of sequence
Backwards:
Newly synthesised strand forms loop and replication moves backwards - expansion by one extra repeat on that strand

Opposite for when template strand slips - deletion on that strand

Expansion of VNTR can worsen some genetic diseases (e.g. huntingtons CAG repeats progressively disrupting HTT gene)

34
Q

DNA fingerprinting

A

Extract dna from blood skin hair follicle etc
Digest dna with restriction enzymes
Separate by gel electrophoresis
Transfer to membrane (southern blot) and incubate with probe that sticks to complementary mini satellite fragments
Pattern of bands gel makes specific barcode

Can alternatively amplify them with PCR instead so less dna is needed
Each pcr amplifys different microsattelites and tags them with different fluorophore

35
Q

Epigenetics!

A

Mitotically Heritable info on top of dna sequence
Explains difference in trusts due to effects of external cues (environment, diet…) on gene expression

Allows different cell types with different expression of the same genome
Allows cell differentiation
As Pluripotent stem cells differentiate ensures they are locked into that cell lineage as you don’t want eg a Nueron to lose its identity

36
Q

Chromatin landscape

A

Epigenetic changes are cell type specific
Allows
Expression of one set of genes
Repression of other genes

Epigenime can be established by packing dna into different chromatin states

37
Q

Chromatin packing structure?

A

Nucleosome - dna wrapped around histone
Packing of dna into nucleosome blocks access to replication/transcription machinery - closed chromatin confirmation

Loosely packed nucleosimes allows access to genes - open chromatin containing active genes

38
Q

Histone code?

A

Controls access to genes
Repressive marks:
H3K9 trimethylation
H3K27 trimethylation

Active marks:
Enhancer histone marks-
H3K27 acetylation
H3K4 (di)methylation
Allow enhancers of transcription to bind
Promoter histone marks-
H3K4 trimethylation
Allow transcription machinery to bind

39
Q

Heyerochromatin subtypes?

A

Constitutive:
10% of genome
Gene poor, inactive
Replicates late in S phase
ALWAYS heterochromatin

Facultative:
Shares features with euchromatin
Replicated early in a phase
Genes are silenced and inactive
Though different genes here are active in different cells
So are part of genome where cell type specific Epigenetic and gene expression differences are

40
Q

multi scale Organisation of chromatin?

A

Dna around histone octamer

Nucleosiomes come together as clutches (2kb)

Form into chromatin fibres (loops or nanodomains 10-100kb)

Fibres part of larger Topologically Associating Domains held together with cohesin and CTCF (a few megabases)

Domains come together into:
Compartment A -for active euchromatin
Compartment B for heterochromatin

All of these organise into separate chromosome territories

41
Q

Nucleosome internal structure?

A

Octamer
1 H3 H4 tetramer
2 H2A H2B dimers

146 boo of dna wrapped around it

Linker histone H1 can wrap the linker DNA causing the Nucleosome to wrap another 45bp
Usually accessible linker dna is now less accessible
Forms zig zag chromatin fibre or unstructured globule (clutch)

42
Q

Histone modification?

A

N and C terminal tails stick out from histones
Modifications switch genes on and off and communicate chromatin state

Lysine acetylation
Methylation of lysine (K) or arginine (R)
Serine phosphorylation
Lysine ubquitination

Most occur in H3 and H4 at N terminal tail
Done by enzymes -
readers bind modifications
writers add modifications
and erasers remove them

Don’t interact directly with dna
Recruited to regulatory elements

43
Q

Histone acetylation?

A

Done to lysine K
Histone acetyltransferases write (HAT)
Histone deacetylases erase (HDAC)

Neutralises K’s positive charge
Weakens interactions between histones and negatively charged phosphate of DNA backbone

Loosens chromatin and triggers transcription

44
Q

Histone methylation?

A

Methyl groups added
Mono di or tri methylated
Done to specific H3 and H4 Ks and Rs

Action is chromatin dependent
Can activate

Or deactivate: recruit chromatin tightening proteins

45
Q

ChIP

A

Chromatin immunoprecipitation
Nuclease or physically fragment dna
Still maintain Nucleosome structure
Ab against specific Nucleosome mark (E.g. H3K9 me3 mark)
Attached to bead
Pull down bead
Enriches for histone mark of interest
Can quantify with qPCR compare how rich regions are in that mark
Purify Dna get rid of histones
Can see what dna is at that mark

46
Q

Kinetochore attraction

A

Heterochromatin centromeres contain satellite repeats
In mitosis kinetochore assembles at centromere
DNA sequence not sufficient to attract it
Special CENP-A (variant of H3) histones in centromere chromatin
Difference in N terminal tail
This is what helps attract kinetochore

47
Q

Position effect variegation?

A

PEV
Section of drosophila eye with w+ gene on and some with it off

Radiation causes inversion of chromosome segment with w+ gene placing it closer to centromere and pericentromeric chromatin

During early embryo development centromeres heterochromatin has spread and hence silenced the w+ gene
This cell divided to form part of eye forming white patch

Only spreads far enough in some cells so still red in some places

Gene silencing can occur through gene relocation
Also gene silencing can be inherited into daughter cells

48
Q

Su(Var)?

A

Suppression of Variegation genes
(Actually cause the variegation - named after mutant ig)
Suvar 3-9:
Is a h3k9 me3
Methylates histones and spreads heterochromatin from centromere

Heterochromatin protein 1
HP1
Is a reader instead of writer
Binds the H3K9 me3 and helps suvar spread heterochromatin

Activity of these increased = more white eye

49
Q

DNA methylation of CpG nucleotides

A

Me group added to cytosines preceding guanines (5’-3’)
Both strands are methylated
Repressive effect

Sequence CG in genome is underrepresented in genome because methylation is mutagenic leading to deamination of 5-methyl cytosine to a thymidine
Overrepresentation of TpG

50
Q

CpG islands?

A

Start sites of genes more enriched with CpG (CpG islands) - these are NOT METHYLATED TO PREVENT TpG conversion
Indicate open chromatin state
Areas of low CpG tend to be highly methylated and heterochromatic as methylation reduces CpG frequency
MeCP2 also recognise methylated CoG and tighten chromatin

51
Q

When is dna methylation established?

A

Early in development
Is hard to remove
Very stable
CpG islands in promoters stay unmethylated
Bound by readers (TFs) that h3K4 me3 - activation marker

52
Q

Gene dosage compensation - X chromosome inactivation

A

Need only one X chromosome to be active
One is randomly inactivated forming a Barr body

X inactivation centre (Xic) locus on X chromosome contains many genes
XIST gene only transcribed on inactivated X
Product is non coding RNA
Binds to inactive X - in cis inactivation
It’s CpG islands also become methylated make sure genes are not reactivated
XIST is not methylated as it needs to be transcribed to inactivate

Inverse on active X
Xist CpG island methylated
Other ones remain unmethylated

53
Q
A

MOG3 (10 decks)

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