Barnes (Eukaryotic Genome Architecture) Flashcards
What is the C value?
- amount DNA in haploid nucleus for given species
What is the C value paradox?
- complexity of organisms doesn’t necessarily correspond to genome size
What is the main cause of differences in genome size?
- protein coding seqs
How dense is the human genome compared to yeast, and why?
- much less densely packed
- as many more introns per gene and genome wide repeats
Why are complex organisms able to have such long introns?
- longer cell division, so no selective pressure
What is satellite DNA, and the different types?
= 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
Why are there differences in satellite DNA between individuals?
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
How can satellite DNA w/in genes cause problems?
- 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
How can satellite DNA be used for DNA fingerprinting?
- 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
How can PCR used for identification of DNA?
- 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
How can RFLPs be used for paternity testing?
- 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
What are the 2 families of transposons?
- cut and paste = DNA transposons (doesn’t generally change genome sizes)
- copy and paste = RNA transposons (use RNA intermediates and increase genome size)
What are transposable elements?
- DNA seq that can change its position w/in genome, sometimes creating or reversing mutations and alt cells genome sizes
What is the structure of a DNA transposon?
- 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
What is transposase, and its role?
- 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
How does insertion of transposons by transposase create direct repeats?
- 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)
How does transposition at S phase increase copy no. of DNA transposons?
- 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
What are the Ac/Ds transposons in maize?
- Ac = activator –> autonomous, has own transposase gene
- Ds = dissociation –> nonautonomous, needs to use transposase enzyme from Ac, same inverted repeat seqs as Ac
What are P elements in Drosophila?
- 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
What happens when P+ and P- strains are crossed?
- 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
What is a retrotransposon?
- transposons that jump via RNA intermediate
- copy and paste mechanism
What are LTR retrotransposons?
- 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
What is the mechanism for LTR retrotransposon transposition?
- 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
How are LTR retrotransposons closely related to RNA viruses?
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
What is an eg. of LTR transposon in mammals?
- endogenous retrovirus
- 8% of human genome
- often only LTR left and rest of transposon lost
What is an eg. of LTR transposon in yeast?
- Ty elements
- 35 copies of Ty1 in haploid yeast genome
What is the structure of long interspersed elements (LINEs)? (non-LTR transposons)
- TSDR -> AT rich region -> ORF1 -> ORF2 -> AT rich region -> TSDR
- ORF1 = RNA binding protein
- ORF2 - reverse transcriptase and DNA endonuclease
What LINEs are present in the human genome?
- L1, L2, L3 = 21% human genome
- only L1 ever still functions
What is the mechanism of LINE transposition?
- 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
What are short interspersed elements (SINEs)? (non-LTR transposons)
- 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
How often does transposition happen?
- not often (due to cellular defense mechanisms)
- 1 transposition in 100s of cell gens
- transposition events will only be fixed if in germline
Are transpositions important in shaping genome?
- yes, over evolutionary timeq
Do all genomes contain same types of transposons?
- no, diff genomes contain diff types of transposons
- diff ratios of retrotransposons to DNA transposons
What are 2 evolutionary consequences of transposition?
- exon shuffling
- gene duplication
How can transposition cause exon shuffling?
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
How can transposition cause gene duplication?
- replication slippage (misalignment and genes missed out or displaced)
- unequal crossing over (genes duplicated)
- retrotransposition of mRNA
Why is gene duplication a common consequence of transposition?
- less than half genes in multicellular euks are “solitary genes”
What are the possible fates of a tandemly repeated duplicated gene?
- 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
What is a pseudogene?
- gene that has lost ability to code for functional protein
What causes a pseudogene?
- 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
What is a common consequence of gene duplication?
- 1 copy stays functional and other degenerates and becomes pseudogene
What is a processed pseudogene?
- 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
What are the 2 diff classes of homologues?
- 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
What are the poss fates of duplicated gene?
- 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
What happened in the globin gene family that is an example of gene duplication events?
- 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
By what mechanism did the globin gene family evolve?
- unequal CO between 2 transposons
- chromosome w/ 2 β- globin genes passed on in germline
- 2 copies evolve independently to gen paralogues
Why are diff globin genes used at diff stages of dev?
- foetal Hb has higher affinity for O than adult Hb
- allows O to be passed from maternal blood to foetal blood
How can duplication of whole genomes occur?
- 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*
In what type of organisms does duplication of whole organisms occur?
- plants
How can polyploidy events become fixed in a pop?
- 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
What have phylogenetic studies revealed about genome duplications in vertebrate linkage?
- 2 poss genome duplication in evo vertebrates
What are HOX genes, and are they an example of genome duplication followed by evo of duplicated genes?
- 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
What are centromeres?
- specialised chromosomal region upon which kinetochores assemble and direct equal segregation of chromosomes during mitosis and meiosis
What are kinetochores?
- structures that link centromeres to spindle MTs
Are chromatin structure and specific DNA seqs conserved in centromeres?
- chromatin structure is and maintained throughout cell cycle
- specific DNA seqs not
What is the process from DNA seq to segregation at mitosis and meiosis?
- DNA seq
- specialised nucleosomes (heterochromatin)
- kinetochore binding
- MT recruitment
- segregation at mitosis and meiosis
What are the characteristics of the yeast (cerevisiae) “point” centromere
- v highly conserved
- v AT rich region II
- only 120bp suffices to direct MT attachment and mitotic segregation
What are the characteristics of the human “regional” centromere?
- 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
What are centromeric nucleosomes?
- 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
How do centromeric nucleosomes vary in humans?
- 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
What is the role of kinetochores?
- recognises centromeric epigenetic markers, eg. alt histones and methylation
- attaches centromere to MTs, allowing segregation at mitosis
What is 1 important part of kinetochore?
- Ndc80 complex
What are holocentric chromosomes?
- eg. in C. elegans
- cenH3 (version of CENP-A) histones distributed throughout chromosome and attach to kinetochore “holocentric” = attachments along whole chromosome
How do origins or rep vary in E. Coli and humans?
- single origin in E. Coli and 10,000s in humans
How are origins or rep in E. Coli and humans similar?
- bidirectional rep forks
- bps broken apart and 2 rep forks formed moving away from each other, each w/ lagging and leading strand
What occurs during early stages of euk rep?
- binding of origin recognition complex
- triggers assembly of pre-rep complex = MCM proteins, CDC6 etc.
- initiation of rep
What are autonomous rep seqs (ARS)?
- rep origins in lower euks
What is the role of ARS in S. cerevisiae?
- 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
What is the role of ARS in S. pombe?
- AT rich intergenic regions
- at least 1/2 intergenic regions have capacity to serve as origins of rep
What are some features often seen in animal rep origins?
- sequence = AT rich, CpG islands
- structure = DNA topology, loop MAR
- chromatin = nucleosome, DNase 1-sensitive site
- transcrip = promoter, enhancer or insulator, start site level
What could diff combos of features seen in animal rep origins determine?
- use of diff origins in diff conditions
- or could change throughout dev
Is there a consensus seq for metazoan origins of rep?
- none found
What are the diff classes of origins of rep?
- 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
How do you find origins or rep - for simple origins?
- 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
How do you find origins of rep - in animal?
- 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
What are telomeres?
- regions at ends of chromosomes consisting of no. of repeats w/ 3’ overhangs
Why are telomeres needed?
- lagging strand req RNA primer which is later removed
- gap filled in from adj Okazaki fragment
- not poss at end of linear chromosome
Is telomere seq well conserved?
- yes
- Tetrahymena = TTGGGG
- all mammals = TTAGGG
Do the no. of repeats in telomeres vary?
- yes
- few repeats in Tetrahymena
- approx 400 in Saccharomyces
- 10-15kb in humans
Why do cells need a mechanism to differentiate end of chromosome from ds break?
- avoid chromosomes being “repaired” and stuck together
- eg. by NHEJ repair pathway
What is the “shelterin” complex?
- no. specialised proteins that bind telomere DNA and each other
- inc TRF1, TRF2, TRF3
What is the role of shelterin complex forming cap on telomere?
- differentiate it from DNA breaks
- promote formation of special 3° structure in DNA => t-loop
- recruit telomerase
- protect from nucleases
What is the protein component of telomerase, and what is its role?
- telomeric reverse transcriptase
- provides template for reverse transcriptase activity
When is telomerase activated?
- when telomere length falls below threshold
What is the mechanism of telomerase?
- 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
What is the “mitotic clock”, and why does it happen?
- 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
What could speed up telomerase shortening?
- some evidence that oxidative stress can –> build up of ss breaks
Why is mitotic clock crucial to ageing?
- cells sentenced to death after certain no. of divisions
What do shorter telomeres mean?
- tend to correlate w/ ageing related disease
Can overexpression of telomerase prevent ageing?
- MAY do in mice
Why is there elevated telomerase activity in 90% cancer cells?
- accum mutations that allow them to become immortal
- often reactivated to allow cont division (2° mutation)
Where are transcriptionally active and silent regions of euk chromosomes found?
- 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
What does G-banding stain?
- transcriptionally silent, AT rich genome regions
How is G-banding carried out?
- metaphase spread of condensed mitotic chromosomes
- treat w/ trypsin to remove proteins
- stain w/ Giemsa
How is chromosome painting carried out using FISH?
- metaphase chromosomes hybridised to fluorescently labelled DNA probes that are specific to seqs in each chromosome (each probe slightly diff colour)
- identification of chromosomes
What can chromosome painting w/ FISH be used for?
- use of any chromosomal rearrangements as diagnostic tool –> see if eg. chunk moved around
- to study evo of chromosomes
