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MBB267 > Barnes (Eukaryotic Genome Architecture) > Flashcards

Barnes (Eukaryotic Genome Architecture) Flashcards

1
Q

What is the C value?

A
  • amount DNA in haploid nucleus for given species
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2
Q

What is the C value paradox?

A
  • complexity of organisms doesn’t necessarily correspond to genome size
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3
Q

What is the main cause of differences in genome size?

A
  • protein coding seqs
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4
Q

How dense is the human genome compared to yeast, and why?

A
  • much less densely packed

- as many more introns per gene and genome wide repeats

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5
Q

Why are complex organisms able to have such long introns?

A
  • longer cell division, so no selective pressure
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6
Q

What is satellite DNA, and the different types?

A

= tandem repeated seqs of 1-500bp , approx 5% of human genome, esp important at mammalian centromeres

  • microsatellites –> mostly 1-4bp and <150 repeats
  • minisatellites –> tandem arrays of 1-5kb around genome
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7
Q

Why are there differences in satellite DNA between individuals?

A

Replication slippage:

  • dissociation of DNA pol during rep
  • nascent DNA strand can rehybridise w/ another repeat in array in misaligned way (w/ repeat earlier or later in array)
  • rep cont but new strand diff length so yields daughter strand longer or shorter than template

Unequal crossing over during meiosis:

  • CO between misaligned repeats on sister chromatids
  • 1 gamete w/ more copies of repeat and 1 w/ less
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8
Q

How can satellite DNA w/in genes cause problems?

A
  • trinucleotide repeat CAG in Huntingtin gene
  • proteins w/ expanded CAG repeats degraded into toxic fragments that accum in neurons and stop them working properly
  • 40+ repeats and affected
  • 36+ and 50% risk to offspring
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9
Q

How can satellite DNA be used for DNA fingerprinting?

A
  • uses restriction fragment length polymorphisms (RFLPs) in minisatellite length between individuals to identify them
  • extract DNA
  • digest w/ convenient RE
  • separate fragments on agarose gel
  • S blot using microsatellite seq as radioactive probe
  • observe characteristic bands for each indiv
  • do this for no. minisatellite seqs to build up profile for identification
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10
Q

How can PCR used for identification of DNA?

A
  • amplified fragment length polymorphism (AFLP)
  • PCR using primers annealing to conserved seq on either side of microsatellite tandem array
  • visualise PCR products in agarose gel
  • or seq DNA
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11
Q

How can RFLPs be used for paternity testing?

A
  • on S blot child has bands which are caused half by father and half by mother
  • more than 2 band as looking at same minisatellites present at many diff loci in genome
  • some alleles present in mother/father and not child as only half inherited from each
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12
Q

What are the 2 families of transposons?

A
  • cut and paste = DNA transposons (doesn’t generally change genome sizes)
  • copy and paste = RNA transposons (use RNA intermediates and increase genome size)
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13
Q

What are transposable elements?

A
  • DNA seq that can change its position w/in genome, sometimes creating or reversing mutations and alt cells genome sizes
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14
Q

What is the structure of a DNA transposon?

A
  • direct repeat on either end = same seq repeated, gen from host genome during transposition
  • inverted repeat inside these = seq plus its reverse complement, transposon recognition site
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15
Q

What is transposase, and its role?

A
  • transcribed and translated by host machinery
  • binds to inverted repeats
  • cuts DNA to remove transposon from its original genomic location
  • creates break at target site to allow transposon to be inserted at new location
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16
Q

How does insertion of transposons by transposase create direct repeats?

A
  • some transposons have preference for target site seq, others insert at random
  • transposase makes staggered cuts in target DNA
  • transposon DNA inserts at target site
  • gaps in target site DNA filled by host repair enzymes
  • gen of direct repeats at insertion site (remain after transposon moved again)
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17
Q

How does transposition at S phase increase copy no. of DNA transposons?

A
  • 1 copy of transposon before S phase
  • S phase = DNA rep and DNA transposition
  • after S phase 1 daughter molecule has 2 copies of transposon
  • builds up over evolutionary time
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18
Q

What are the Ac/Ds transposons in maize?

A
  • Ac = activator –> autonomous, has own transposase gene

- Ds = dissociation –> nonautonomous, needs to use transposase enzyme from Ac, same inverted repeat seqs as Ac

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19
Q

What are P elements in Drosophila?

A
  • DNA transposons found in many modern wild Drosophila (P+), but not in lab strains (mainly descendants from Morgans labs (P-)
  • must have arisen in wild pops since early 20th century
  • when left unchecked P elements transpose at v high rates and lead to severe problems in offspring (= v high mutation rates and infertility)
  • silencing mechanism in cyto limits transposon movement in P+ strains, but not P- strains
  • in new embryo cyto comes from egg
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20
Q

What happens when P+ and P- strains are crossed?

A
  • male P- and female P+ = silencing established in egg cyto, no transposition and successful cross
  • female P- and male P+ = no silencing from cyto of P- egg, but P elements present in paternal DNA, high transposition and unsuccessful cross
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21
Q

What is a retrotransposon?

A
  • transposons that jump via RNA intermediate

- copy and paste mechanism

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22
Q

What are LTR retrotransposons?

A
  • long terminal repeat transposons
  • target site direct repeats -> LTRs -> gag -> pol -> LTRs -> target site direct repeats
  • pol encodes 3 protein activities req for transposition = reverse transcriptase, RNAse H, integrase
  • LTRs important for reverse transcrip mechanism
  • direct repeats gen upon integration
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23
Q

What is the mechanism for LTR retrotransposon transposition?

A
  • gen of RNA molecule and protein products by host pol machinery
  • complex reverse transcriptase mechanism involving retrotransposon reverse transcriptase and RNAseH to prod DNA molecule
  • transport of dsDNA into genome w/ creation of target site direct repeats
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24
Q

How are LTR retrotransposons closely related to RNA viruses?

A

RNA viruses and some LTR transposons:

  • TSDR -> LTR -> gag -> pol -> env -> LTR -> TSDR
  • gag and env encode proteins to make infective virion
  • gag and sometime env conserved in some LTR retrotransposons
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25
Q

What is an eg. of LTR transposon in mammals?

A
  • endogenous retrovirus
  • 8% of human genome
  • often only LTR left and rest of transposon lost
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26
Q

What is an eg. of LTR transposon in yeast?

A
  • Ty elements

- 35 copies of Ty1 in haploid yeast genome

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27
Q

What is the structure of long interspersed elements (LINEs)? (non-LTR transposons)

A
  • TSDR -> AT rich region -> ORF1 -> ORF2 -> AT rich region -> TSDR
  • ORF1 = RNA binding protein
  • ORF2 - reverse transcriptase and DNA endonuclease
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28
Q

What LINEs are present in the human genome?

A
  • L1, L2, L3 = 21% human genome

- only L1 ever still functions

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29
Q

What is the mechanism of LINE transposition?

A
  • transposon transcribed and translated by host machinery, polyadenylated
  • ORF1 protein binds LINE RNA, ORF 2 binds LINE polyA in cyto
  • RNA transported into nucleus
  • ORF2/polyA binds to complementary polyT DNA seq somewhere in genome
  • endonuclease activity from ORF2 nicks DNA at staggered sites
  • ORF2 reverse transcriptase activity primed by host DNA seq
    • -> RNA acts as template
    • ->often reverse transcrip doesn’t reach end so transposon truncated
  • ORF2 cont synthesis, using host DNA as template
  • 2nd strand of DNA made by host enzymes
    • -> direct repeats gen due to staggered cut at insertion repeat
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30
Q

What are short interspersed elements (SINEs)? (non-LTR transposons)

A
  • nonautonomous, req enzymes from LINEs to function
  • AT rich seqs that bind to ORF1 and ORF2
  • Alu element
    • -> common in primate genomes, over 1 mil copies in human genome but many are truncated
    • -> consensus seq = 282 bp
    • -> named as contain Alu restriction site
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31
Q

How often does transposition happen?

A
  • not often (due to cellular defense mechanisms)
  • 1 transposition in 100s of cell gens
  • transposition events will only be fixed if in germline
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32
Q

Are transpositions important in shaping genome?

A
  • yes, over evolutionary timeq
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33
Q

Do all genomes contain same types of transposons?

A
  • no, diff genomes contain diff types of transposons

- diff ratios of retrotransposons to DNA transposons

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34
Q

What are 2 evolutionary consequences of transposition?

A
  • exon shuffling

- gene duplication

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35
Q

How can transposition cause exon shuffling?

A

1) - due to COs between transposons in diff parts of genome
- exons of diff genes both flanked by same transposon swapped over when there is recombination in transposon seq
2) - due to mistakes in transposition
- exon in between may be excised from genome and inserted at new location
- LINE transposon might use polyA signal of neighbouring gene instead of own, so adds exon onto its normal transcript

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36
Q

How can transposition cause gene duplication?

A
  • replication slippage (misalignment and genes missed out or displaced)
  • unequal crossing over (genes duplicated)
  • retrotransposition of mRNA
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37
Q

Why is gene duplication a common consequence of transposition?

A
  • less than half genes in multicellular euks are “solitary genes”
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38
Q

What are the possible fates of a tandemly repeated duplicated gene?

A
  • all copies conserved as v high amounts RNA need to be transcribed so beneficial
  • non transcribed spacer seqs in between can be quite divergent but genes relatively well conserved
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39
Q

What is a pseudogene?

A
  • gene that has lost ability to code for functional protein
40
Q

What causes a pseudogene?

A
  • accum mutations that prevent gene functioning

- if no selection pressure due to presence of 2nd copy then more mutations accum and gene function lost alltogether

41
Q

What is a common consequence of gene duplication?

A
  • 1 copy stays functional and other degenerates and becomes pseudogene
42
Q

What is a processed pseudogene?

A
  • another effect of LINE transposons on genome
  • gen by reverse transcrip of functional mRNA and insertion of cDNA into genome by LINE proteins
  • these inserted seqs don’t have proper processing signal so generally not functional
43
Q

What are the 2 diff classes of homologues?

A
  • orthologues = evolved by speciation, same gene in 2 diff species, evolved separately after divergence of species
  • paralogues = evolved by duplication, in same species, gene duplicated then diffs accum in 2 copies
44
Q

What are the poss fates of duplicated gene?

A
  • pseudogene
  • neofunctionalisation = 1 copy gains new function (by chance could be beneficial and become fixed)
  • subfunctionalisation = each copy specialises (eg. when protein w/ 2 domains w/ diff functions, OR to adapt to diff circumstances
45
Q

What happened in the globin gene family that is an example of gene duplication events?

A
  • human Hb has 2 α-family and 2 β-family chains
  • vertebrate globin gene family contains no. of members, gen by gene duplication, that have evolved to have slightly diff properties = subfunctionalisation
  • α and β diverged but still recognisable homologues –> could be silent mutations, can use seq diffs to construct phylogenetic tree of family members
  • gene duplication and subfunctionalisation
  • evolved into myoglobin, α-globins and β-globins
  • at least 80% between diff β-globin genes as diverged more recently
46
Q

By what mechanism did the globin gene family evolve?

A
  • unequal CO between 2 transposons
  • chromosome w/ 2 β- globin genes passed on in germline
  • 2 copies evolve independently to gen paralogues
47
Q

Why are diff globin genes used at diff stages of dev?

A
  • foetal Hb has higher affinity for O than adult Hb

- allows O to be passed from maternal blood to foetal blood

48
Q

How can duplication of whole genomes occur?

A
  • 2n gametes can be gen by meiotic nondisjunction
  • can combine w/ 1n gametes to form 3n embryo, or w/ another 2n gamete to form 4n embryo
  • organisms w/ even no. n tend to be more stable, although errors in mitosis and meiosis more likely
  • DIAGRAM*
49
Q

In what type of organisms does duplication of whole organisms occur?

A
  • plants
50
Q

How can polyploidy events become fixed in a pop?

A
  • 2 copies present so less selective pressure
  • can allow for divergence and specialisation of 2 copies
  • over evolutionary time lots of duplicated material lost by mutation or deletion = diploidisation, ie. returning back to diploid state
51
Q

What have phylogenetic studies revealed about genome duplications in vertebrate linkage?

A
  • 2 poss genome duplication in evo vertebrates
52
Q

What are HOX genes, and are they an example of genome duplication followed by evo of duplicated genes?

A
  • TFs that determine anterior-posterior axis of animal dev
  • complicated system of human genes involved due to genome duplication events creating redundancy
  • 4 copies of each gene in mammals compared to ancestral genes
53
Q

What are centromeres?

A
  • specialised chromosomal region upon which kinetochores assemble and direct equal segregation of chromosomes during mitosis and meiosis
54
Q

What are kinetochores?

A
  • structures that link centromeres to spindle MTs
55
Q

Are chromatin structure and specific DNA seqs conserved in centromeres?

A
  • chromatin structure is and maintained throughout cell cycle
  • specific DNA seqs not
56
Q

What is the process from DNA seq to segregation at mitosis and meiosis?

A
  • DNA seq
  • specialised nucleosomes (heterochromatin)
  • kinetochore binding
  • MT recruitment
  • segregation at mitosis and meiosis
57
Q

What are the characteristics of the yeast (cerevisiae) “point” centromere

A
  • v highly conserved
  • v AT rich region II
  • only 120bp suffices to direct MT attachment and mitotic segregation
58
Q

What are the characteristics of the human “regional” centromere?

A
  • alphoid satellite DNA –> AT rich seqs, each repeat is 17bp
  • in tandem arrays at centromeres of all human chromosomes
  • higher order structure of several repeats w/ slightly divergent seqs –> forms larger repeating unit
59
Q

What are centromeric nucleosomes?

A
  • specific and highly conserved
  • standard nucleosome comprises 8 histones, inc 2x H3
  • CENP-A replaces H3 at euk centromere
    • -> specialised histone that marks nucleosome as diff
    • -> CENP-A dictates kinetochore binding
60
Q

How do centromeric nucleosomes vary in humans?

A
  • additional mod nucleosomes at human centromeres –> H2A.Z integrated instead of H2 and H3 methylated
  • around centromere, repression of transcrip, “pericentric heterochromatin” –> specific methylation of histones at these nucleosomes
61
Q

What is the role of kinetochores?

A
  • recognises centromeric epigenetic markers, eg. alt histones and methylation
  • attaches centromere to MTs, allowing segregation at mitosis
62
Q

What is 1 important part of kinetochore?

A
  • Ndc80 complex
63
Q

What are holocentric chromosomes?

A
  • eg. in C. elegans
  • cenH3 (version of CENP-A) histones distributed throughout chromosome and attach to kinetochore “holocentric” = attachments along whole chromosome
64
Q

How do origins or rep vary in E. Coli and humans?

A
  • single origin in E. Coli and 10,000s in humans
65
Q

How are origins or rep in E. Coli and humans similar?

A
  • bidirectional rep forks

- bps broken apart and 2 rep forks formed moving away from each other, each w/ lagging and leading strand

66
Q

What occurs during early stages of euk rep?

A
  • binding of origin recognition complex
  • triggers assembly of pre-rep complex = MCM proteins, CDC6 etc.
  • initiation of rep
67
Q

What are autonomous rep seqs (ARS)?

A
  • rep origins in lower euks
68
Q

What is the role of ARS in S. cerevisiae?

A
  • 11 bp consensus seq
  • 250-400 rep origins in each round of rep
  • only some actually initiate rep –> essential but not sufficient for origin activity
  • transcriptionally silent areas more likely to be bound by rep proteins
  • only subset of 100s of ARS used –> variable subset selected to some extent, but some used more often than others
69
Q

What is the role of ARS in S. pombe?

A
  • AT rich intergenic regions

- at least 1/2 intergenic regions have capacity to serve as origins of rep

70
Q

What are some features often seen in animal rep origins?

A
  • sequence = AT rich, CpG islands
  • structure = DNA topology, loop MAR
  • chromatin = nucleosome, DNase 1-sensitive site
  • transcrip = promoter, enhancer or insulator, start site level
71
Q

What could diff combos of features seen in animal rep origins determine?

A
  • use of diff origins in diff conditions

- or could change throughout dev

72
Q

Is there a consensus seq for metazoan origins of rep?

A
  • none found
73
Q

What are the diff classes of origins of rep?

A
  • flexible = used sometimes, randomly
  • constitutive = always used (this is minority)
  • inactive = never used under normal conditions, used in eg. stressful conditions when need quick rep
74
Q

How do you find origins or rep - for simple origins?

A
  • eg. autonomous rep assays
  • clone pieces of S. cerevisiae genome into recombinant plasmids to test whether they could guide rep (w/ drug resistance marker)
  • test many diff seqs to find ones that allow plasmids to be rep
  • no good for more complex sites in higher euks
75
Q

How do you find origins of rep - in animal?

A
  • eg. studies on nascent strand abundance
  • can isolate newly synthesised DNA using BrdU
  • BrdU is thymidine analogue
  • can be immunoprecipitated
  • add BrdU to medium instead of thymidine, allows you to pull down nascent DNA
  • then microarrays or high throughput seq to identify which parts of genome those are
76
Q

What are telomeres?

A
  • regions at ends of chromosomes consisting of no. of repeats w/ 3’ overhangs
77
Q

Why are telomeres needed?

A
  • lagging strand req RNA primer which is later removed
  • gap filled in from adj Okazaki fragment
  • not poss at end of linear chromosome
78
Q

Is telomere seq well conserved?

A
  • yes
  • Tetrahymena = TTGGGG
  • all mammals = TTAGGG
79
Q

Do the no. of repeats in telomeres vary?

A
  • yes
  • few repeats in Tetrahymena
  • approx 400 in Saccharomyces
  • 10-15kb in humans
80
Q

Why do cells need a mechanism to differentiate end of chromosome from ds break?

A
  • avoid chromosomes being “repaired” and stuck together

- eg. by NHEJ repair pathway

81
Q

What is the “shelterin” complex?

A
  • no. specialised proteins that bind telomere DNA and each other
  • inc TRF1, TRF2, TRF3
82
Q

What is the role of shelterin complex forming cap on telomere?

A
  • differentiate it from DNA breaks
  • promote formation of special 3° structure in DNA => t-loop
  • recruit telomerase
  • protect from nucleases
83
Q

What is the protein component of telomerase, and what is its role?

A
  • telomeric reverse transcriptase

- provides template for reverse transcriptase activity

84
Q

When is telomerase activated?

A
  • when telomere length falls below threshold
85
Q

What is the mechanism of telomerase?

A
  • RNA component bps w/ 3’ overhang
  • elongation of overhang using RNA as template
  • translocates further out along 3’ overhang
  • elongation of overhang using RNA as template
  • RNA removed and synthesis of 2nd strand by DNA pol using overhang as template
86
Q

What is the “mitotic clock”, and why does it happen?

A
  • single cell euks are “immortal” = high telomerase activity
  • humans have high telomerase activity in stem and germline cells, but low in somatic cells
  • in general telomeres in somatic cells shorten as organism ages
  • telomeres shorter than certain length can’t bind shelterin –> triggers cell cycle arrest, senescence, apoptosis and genome instability
87
Q

What could speed up telomerase shortening?

A
  • some evidence that oxidative stress can –> build up of ss breaks
88
Q

Why is mitotic clock crucial to ageing?

A
  • cells sentenced to death after certain no. of divisions
89
Q

What do shorter telomeres mean?

A
  • tend to correlate w/ ageing related disease
90
Q

Can overexpression of telomerase prevent ageing?

A
  • MAY do in mice
91
Q

Why is there elevated telomerase activity in 90% cancer cells?

A
  • accum mutations that allow them to become immortal

- often reactivated to allow cont division (2° mutation)

92
Q

Where are transcriptionally active and silent regions of euk chromosomes found?

A
  • roughly correspond to gene-rich and gene-poo regions
  • gene-poor tend to be AT rich, w/ lots of repetitious seqs and transcriptionally silent (more closed chromatin structure)
  • regions characterised by diffs in chromatin structure –> histone mods leading to open or closed structure
  • centromeres have specialised chromatin markers
93
Q

What does G-banding stain?

A
  • transcriptionally silent, AT rich genome regions
94
Q

How is G-banding carried out?

A
  • metaphase spread of condensed mitotic chromosomes
  • treat w/ trypsin to remove proteins
  • stain w/ Giemsa
95
Q

How is chromosome painting carried out using FISH?

A
  • metaphase chromosomes hybridised to fluorescently labelled DNA probes that are specific to seqs in each chromosome (each probe slightly diff colour)
  • identification of chromosomes
96
Q

What can chromosome painting w/ FISH be used for?

A
  • use of any chromosomal rearrangements as diagnostic tool –> see if eg. chunk moved around
  • to study evo of chromosomes

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