Chromatin Arrangement Flashcards

Question

What proportion of human genome is coding sequences?

Answer 1

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

Answer 2

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

Answer 3

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

Answer 4

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

Answer 5

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

Answer 6

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

Answer 7

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

Answer 8

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)

Answer 9

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)

Answer 10

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

Answer 11

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

Answer 12

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

Answer 13

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

Answer 14

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

Answer 15

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

Answer 16

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

Answer 17

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)

Answer 18

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

Answer 19

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

Answer 20

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

Answer 21

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

Answer 22

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

Answer 23

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

Answer 24

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

Answer 25

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

Answer 26

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

Answer 27

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

Answer 28

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