Jones - How do organisms preserve the integrity of their mt genome? Flashcards
What is the structure of mtDNA?
- ds closed circle
What does human mtDNA encode and how big is it?
- 13 polypeptides
- 22 tRNAs
- 2 rRNAs
- 16.6kb size
What do the features of mtDNA inc?
- D loop = displacement loop (or control region)
- HSP = heavy strand promoter region
- LSP = light strand promoter region
- OH = H strnad origin of rep
- OL = L strand origin of rep
Does mammalian mtDNA have non coding DNA?
- no introns
- but some area of non coding DNA, eg. D loop, OL
How do mts increase SA, and why is this needed?
- cristae
- for oxphos
Do mts always act independently, why?
- fuse together to form diverse networks t/o cell
- do this at diff stages t/o cell cycle
What is meant by intergenomic communication?
- transcrip and translation of mtDNA is dependant of effective interaction with nucleus
How much does mt genome vary in size between species?
- 16-18kb in mammals
- 75kb in yeast
- up to 400kb in plants
Why does mt genome size vary between species?
- presence and sizes of introns
- also considerable difference in genes encoded, reflecting many changes since endosymbiotic event (movement of genes/loss of genes if redundant etc.)
Why does mtDNA have a v variable copy no?
- multiple genomes per organelle, multiple organelles per cell
How does copy no. vary between somatic and germline cells?
- 20-200 in somatic cells
- over 200,000 in mature oocyte
Where does mtDNA originate from, what is the evidence from this?
- bacterial origins
- mt ribosomes differ
- variation in triplet code (eg. UGA usually stop, but in mtDNA encodes Trp)
What are the diff complexes of ETC?
- Complex I = NADH deHase
- Complex II = succinate deHase
- Complex III = cytochrome c reductase
- Complex IV = cytochrome c oxidase
- Complex V = ATP synthase
Are any of the ETC complexes entirely nuclear encoded?
- only complex II
What is the consequence of the fact that vital cellular apparatus is encoded by 2 genomes?
- need effective communication between them for normal cellular function
What are the features of mtDNA rep?
- 2 origins of rep: OH and OL –> most genes encoded by H, few by L
- transcrip commences from HSP and LSP
mtDNA rep, dep on transcrip of short section from LSP - TFs, cofactor and pols all nuclear encoded → need these factors to come together and interact for effective rep
What is TFAM?
- essential mt transcrip factor
What is special about POLG?
- DNA pol that is only mitochondrial
What was an experiment which showed TFAM was essential for maintaining mtDNA copy no. and for ETC function?
- actin is control
- w/o TFAM mtDNA can’t be maintained (in homozygous KO) –> shown by PCR
- levels TFAM less in heterozygote
- TFAM +/- = reduced mtDNA copy no., reduced mtDNA transcript and ETC dysfunction in heart
- TFAM -/- = severe mtDNA depletion, abolished OP, enlarged mt, growth retardation, die prior to embryonic day E10.5
How does TFAM reg mtDNA rep?
- binds LSP
- together w/ complex of other factors enables transcrip of entire copy of L stand and short primer for rep (both from LSP)
- get transition from RNA to DNA, POLG recruited
- rep of H strand –> need TFAM to bind before mt rep
What are the roles of TFAM?
- req for transcrip from LSP and HSP –> so essential for mtDNA rep
- levels may directly control mtDNA copy no.
- also important packaging role and most abundant prot in mt nucleoids (another is mtSSB, a mt ss DNA BP)
- nucleoid prots reg stability, rep, transcrip and segregation of mtDNA
What is the structure of TFAM?
- high mobility group box A and B joined by linker dom
- short C-ter dom attached to HMG-box B
- binding recognition site upstream of HSP and LSP
How does TFAM binding to LSP and HSP affect the structure of mtDNA, and why is this important?
- forces mtDNA into U shape
- structurally important for activation of transcrip from these 2 sites
Why might there be problems if have mt and nDNA from diff source (/species) and how might this affect OP?
- if changes in seq (recognition/binding sites) then wont get same binding (more/less effective) → so more/less trancrip
How does nuclear and mtDNA interplay?
- nuclear encoded enz req for mtDNA transcrip and rep
- co-assembly of nuclear encoded and mtDNA encoded subunits of resp chain complexes
- majority of prots that function in mt are nuclear encoded
- over 1000 nuclear encoded products are essential to mt function
What is the effect of crosses between diff species on mtDNA?
- more diverse species = more problems
- some level of cross species compatibility → resp chain defects when crossed mice with rats, but diff species of mouse were fine
How is mtDNA inherited in humans (and most organisms)?
- maternally (or uniparentally) inherited
How are yeast able to survive loss of mt function, and how is there mtDNA inherited?
- facultative anaerobes, so can gen energy through glycolysis
- inherit mtDNA from both parental cells and therefore have heteroplasmic mtDNA pop → but reversion to homoplasmy w/in 20 cell divisions
What causes variation in mtDNA?
- no recomb of parental alleles, unlike nDNA
- faster mutation rate than nDNA = at least 10x
- high level of variation in D loop as non encoding so fewer consequences, known as hypervariable region
Why does mtDNA have a faster mutation rate then nDNA?
- as no protective histones
- lack of proofreading by POLG
- decreased level of repair in mtDNA
What are the sources of heteroplasmy?
- age related muts
- inheritance of germline mut
- introd of foreign mt to reconstructed embryos
What is the mtDNA bottleneck, and what suggests its existence?
- next gen has quite diff pop, so must be taking small no. from parent
- so must be dramatic reduction in no. mtDNA copies at some point in oogenesis to allow these changes to occur
What are the 3 theories for the mtDNA bottleneck?
1) passive reduction of mtDNA
2) packaging into homoplasmic clusters
3) focal rep of mtDNA
What would a passive reduction of mtDNA involve (a mtDNA bottleneck theory)?
- huge copy no. in oocyte, fertilised, then reduced no. copies after each cell division
- so by chance segregating in diff way to cells that spawn next gen
What would packaging into homoplasmic clusters involve (a mtDNA bottleneck theory)?
- passive reduction not enough
- mt genomes cluster in nucleoids or multiple nucliods cluster, and get segregation of nucleoids/groups of nucleoids
What would a focal rep of mtDNA involve (a mtDNA bottleneck theory)?
= segregation then selective amp of certain mtDNA mols
- random segregation v early in dev
- so genomes distrib at random to each of cells, so cells of next gen have much lower mtDNA copy no.
- after puberty get selective amp
What evidence is there for a focal rep of mtDNA?
- study visualised replicating mtDNA in vivo
- after 2 hours not much new mtDNA being made even though lots of TFAM, probs lots of mtDNA just not all being replicated → selective amp of certain mtDNAs during oocyte maturation, so heteroplasmy levels can change dramatically between gens
Why do we not know which theory of mtDNA bottleneck is correct?
- conflicting data on extent of mtDNA copy no. decline in PGCs
- plenty of evidence to show lack of mtDNA rep during early embryogenesis
- multiple sources of experimental evidence that show selection of beneficial mtDNA variants over mutants
- combo of models most likely to be correct
Why is elimination of paternal mtDNA needed?
- to protect subsequent gens from heteroplasmy
What is the dilution effect?
- copy no. of mtDNA in sperm approx 20, comp to 20,000 in oocyte
- reduced during spermatogenesis
What evidence is there for the involvement of TFAM in elimination of paternal mtDNA?
- TFAM and mtDNA levels decline during maturation
- WB data shows that in comp to other tissues testes have lower levels TFAM, and declines t/o puberty → therefore levels of mtDNA are much reduced in mature sperm than spermatogonia and reduction of sperm TFAM levels t/o dev
What mechanism is in place to deal w/ paternal mtDNA that still enters oocyte?
- active degrad of paternal mtDNA in fertilised oocyte
Is homoplasmy ‘normal’?
- yes, it’s the desired state
Is heteroplasmy rare, and when is it more common?
- heteroplasmy not uncommon
- increases w/in indivs w/ age and assoc w/ ageing effects and numerous diseases
How many people carry known disease causing point mutation, comp to those w/ mt disease?
- 1 in 200 carry known disease causing point mutation
- only 1 in 5000 have mt disease
Can mt diseases arise from a nuclear mutation?
- yes, if impairs mt function
What do threshold levels mean in relation to mt disease
- certain level of genomes must be mutated before disease evident (can be up to 90%, if 10% encode enough normal polypeptides for normal levels of oxphos to occur)
How can heteroplasmy lead to homoplasmy through mitotic segregation?
- genomes randomly segregated to each daughter cell → so heteroplasmic cell can prod homoplasmic daughter
- DIAG*
When does random segregation of mtDNA variants occur?
- PGCs made early in dev
- have certain mtDNA pop
- undergo mitosis and meiosis before get mature oocytes
- during cell divisions get random segregation of mutant genomes so eventual pool of oocytes can have v variable mutant load
What do PGCs (primordial germline cells) become?
- sperm/egg cells
How do levels of mutant mtDNAs affect the outcome, and what is the consequence of this?
- low levels mutant mt unlikely to suffer
- high levels more likely to
- so can see how severity of disease can shift rapidly from 1 gen to next and can vary a lot between siblings
Why is there pref rep of normal mtDNA?
- need protective mechanism acting at this point to prevent selective rep of mutated genomes
How was a cell specific oxphos req shown experimentally?
- looked at patients w/ mutation in EFG1 (mt translation factor)
- ran WB against diff ETC complexes
- muscle and heart cells had much higher levels of prot (logical as energy intensive tissues)
- patients had gen less prot than control
- complex II was control, as all nuclear encoded
- so dep on tissue, a mutant load of eg. 90% may/may not be important
How is mt disease spread out across genome?
- every mtDNA encoded subunit of ETC is assoc w/ at least 1 known disease causing mutation
Where is mt disease shown?
- energy intensive tissues
What can mt disease result from?
- mutations in mtDNA, nuclear genes encoding OXPHOS subunits, or proteins req for their translation or assembly
What kind of inheritance do mt disorders caused by nuclear mutations show?
- mendelian
What inheritance pattern do mt disorders caused by mtDNA mutations show?
- maternally inherited
- but follow laws of pop dynamics
What did a study looking a whether disease severity correlated w/ levels of heteroplasmy find?
- looked at A>G mutation in 12S rRNA gene of mtDNA, that causes hearing loss
- age of onset and symptom severity varied
- measured heteroplasmy levels in all in pedigree, by direct seq analysis
- risk of deafness increased w/ increased mutant load, BUT levels of heteroplasmy did not correlate w/ disease severity
- so mutant load v important in determining phenotype, but not only variable
What type of mutations can happen to POLG, and what is most common?
- 94% missense
- low no. fs/dels
- CAG repeats
How can POLG mutations be inherited?
- majority autosomal recessive
- except autosomal dominant progressive external ophthalmoplegia (adPEO)
What do POLG mutations result in?
- array of 2° mtDNA mutations
- deletions (no rep)
- mtDNA depletion
POLG mutations are clinically what?
- heterogeneous
Are mt functions conserved between humans and yeast?
- yes, highly conserved between humans and S. cerevisiae
Why are yeast models used to study mt disease?
- poss to undertake large scale screens
- genetic manipulations easy
- biochemical analyses well established
- facultative anaerobes, so can survive on fermentable C sources in absence of mt function
- growth phenotype simple to assess
- become homoplasmic w/in few gens, so could set up series of mutants w/ diff nuclear background against 1 mt background (or opp)
How was it shown experimentally than 2° nuclear mutations can impact on phenotype?
- looked at growth dynamics under resp conditions for interspecific hybrids
- looked at effects of 4 diff nuclear contexts on growth phenotype of particular mutation
- serial dilutions of yeast cells harbouring single mt mutation, looking at growth of glycerol (no growth suggests OXPHOS not working)
What are 3 methods of assisted reproductive tech?
1) mt supplementation to treat infertility (cyto transfer)
2) mt supplementation/replacement to treat mt diseases caused by mt defects
3) somatic cell nuclear transfer to gen stem cells
What kind of patients is cytoplasmic transfer used to treat?
- infertile
How does cytoplasmic transfer work?
- threshold no. copies of mtDNA genomes for egg to be fertile
- so take some extra ooplasm from another of patients eggs or donor egg and supplement/ transfer some in, so no. mt genomes above threshold, allowing egg to become fertile
Is cytoplasmic transfer successful?
- was initially
- then banned due to safety concerns
Who is mt replacement therapy used for?
- women carrying mt disease, to allow them to have baby w/o disease
What 2 methods can be used to carry out mt replacement therapy?
- PNT (pro nuclear transfer)
- spindle trasnfer
How is spindle transfer carried out?
- start w/ unfertilised patients egg w/ abnormal mt and unfertilised donor egg w/ normal mt
- at fert eggs arrested at metaphase 2 (don’t complete meiosis till after fert) → so all chromosomes clustered on metaphase spindle
- spindle and assoc chromosomes removed as karyoplast from both eggs → discarded from donor egg and patients spindle fused into “enucleated” donor egg (so have donor ooplasm and mtDNA w/ patients chromosomes)
- reconstituted egg fertilised w/ sperm
How is PNT carried out?
- patients and donors egg fertilised and form zygote
- take out pronuclei from donor egg and insert pronuclei from patient egg
What happens to mutant mtDNA pop during mt replacement therapy?
- mt cluster around metaphase 2 spindle
- so in spindle transfer although trying to just take chromosomes and leave all mutant mt, prob transfer of some mutant genomes into final reconstructed oocyte
- do they get diluted out/is there persistence/is there rep?
How could pref rep of mutant mtDNAs be a problem?
- some of children born of cytoplasmic transfer analysed and shown to harbour heteroplasmy
- if ending up w/ eg. 1% of mutant mtDNA genomes, what happens if prefrep and segregate into diff tissues, might go into oocyte and rapidly increase over gens to cause mt disease
- part of debate was whether to only allow for male embryos, but didn’t due to problem of gender selection
What is the reported contrib of donor mtDNA in somatic cell nuclear transfer, and what does this mean?
- 0-63% in embryos
- 0-59% in offspring
- so must be pref rep of donor mtDNA
When is pref rep more likely?
- if species more divergent → less likely to pref rep if species same/more similar
How is persistence overcome in somatic cell nuclear transfer, and what is the problem w/ this?
- mtDNA depletion of donor cells
- prob not approp for oocyte as would be damaging
What did a paper studying small fish discover about how elimination of sperm mtDNA was achieved - main findings?
(Nishimura)
- happened through gradual decrease in no.s of mt nucleoids during spermatogenesis
- and rapid digestion of sperm mtDNA just after fertilisation, achieved by complete destruction of mt structure (poss to avoid transmission of pot deleterious sperm mtDNA to offspring)
What did a paper studying small fish discover about how elimination of sperm mtDNA was achieved - experiments/explanations?
(Nishimura)
- mtDNA nucleoids present in living sperm at every stage during dev (through staining to visualise mtDNA)
- no. nucleoids and copy no. of mtDNA per nucleoid changes during spermatogenesis
(staining to visualise DNA) → 5 fold decrease in no. nucleoids per nucleus, but same mtDNA copy no., so although amount of mtDNA decreases a lot, mtDNA composition of each nucleoid is conserved - active digestion of male mtDNA in natural fertilisation → diluted paternal mtDNA in initial oocyte, but actively degrad before 2 cell stage
- removal of sperm mtDNA in egg and sperm after fertilisation
→ microinjection of sperm mtDNA into egg, stained mtDNA, then used optical tweezers to remove single sperm w/ or w/o mtDNA and analysed w/ PCR
→ saw decrease in levels of mtDNA in sperm after fertilisation
→ saw polymorphism from maternal mtDNA always present in cases where sperm mtDNA may/may not be (shows inheritance of mtDNA only from mother)
What did a paper studying drosophila discover about elimination of sperm mtDNA before fertilisation - main findings?
(DeLuca)
- 2 mechanisms to avoid transmission of sperm mtDNA to zygote
1) endonucleases G degrades nucleoids in sperm, thus removing mtDNA in mt
2) back up mech: actin containing investment cone sweeps all nucleoids
What did a paper studying drosophila discover about elimination of sperm mtDNA before fertilisation - experiments/explanations?
(DeLuca)
- qPCR to quantify paternal mtDNA
→ not detected in zygote immed after fertilisation = less than 1 male mtDNA per egg
→ so mtDNA removed prior to sperm maturation, so none/v little transferred to egg - mtDNA levels followed t/o spermatogenesis and found nucleoids eliminated in coord w/ the sperm elongation phase
- characterising the mechanism through informatics screen for nucleases → found 5 homologous genes (inc EndoG) w/ predicted mt targeting signals, only EndoG mutations prevented nucleoid elimination
What did a paper studying contrib of paternal mtDNA to the next gen in mice find - main findings?
(Rojansky)
- paternal mt doesn’t contrib to next gen and is degrad by mitophagy
- MUL1 and PARKIN have roles in this
What did a paper studying contrib of paternal mtDNA to the next gen in mice find - experiments/explanations?
(Rojansky)
- tracked paternal mt in early mouse embryo, by fluorescence microscopy → after 84 hrs paternal mt almost completely lost, no reduction of maternal mt
- identified mitophagy genes, as exp increased in oxphos inducing conditions → 3 genes identified
oxphos req for paternal mt to be lost - knockdown experiments to identify roles of PINK1, PARKIN and MUL1
→ MUL1 compensated for PARKIN
→ knockdown of both reduced ubiquitination of mt
→ PINK1 knockdown alone reduced ubiquitination - paternal mt deleted through mitophagy → knockdown of mitophagy genes, these genes were req for elimination of paternal mt
- looked at whether paternal and maternal mt fuse, paternal mt didn’t fuse w/ other mt and segregation of paternal mt important for degrad
What did a paper studying the role of autophagy in sperm mtDNA degrad in C. elegans find - main findings?
(Sato)
- autophagy involved in degrad sperm mt immed after fertilisation, so only maternal inheritance of mtDNA
What did a paper studying the role of autophagy in sperm mtDNA degrad in C. elegans find - experiments/explanations?
(Sato)
- measured disappearance of paternal mt and saw male mt initially enter embryo, but degrad by 4 cell stage
- monitored autophagosome formation, induced in 1 cell stage embryo, most disappeared by 16 cell stage, then some reappeared around 32 and 64 cell stage, before disappearing again in late embryo dev → shows dynamic reg of genes involved in autophagosome formation
- induction of autophagosomes dep on fertilisation
→ used Ts mutants against fertilisation, embryogenesis
→ mutants in fertilisation prevented induction of autophagosomes
→ mutants in embryogenesis, but not affecting fertilisation still had induction of autophagosomes around paternal mtDNA - autophagosomes degrade mt → autophagosome and paternal mtDNA signals disappear at same time, so is degrading paternal mtDNA
What did a paper looking at mutations in prot coding genes of mtDNA in mice find - main findings?
(Stewart)
- purifying selection against non-synonymous mutations in prot-coding genes of mtDNA
What did a paper looking at mutations in prot coding genes of mtDNA in mice find - experiments/explanations?
(Stewart)
- looked at ratio of nonsynonymous to synonymous mutations, found purifying selection against nonsynonymous changes in prot coding genes and selection for silent mutations
- comp mutations in 1st and 2nd codon position, to 3rd, decrease in 1st/2nd position as likely to cause change in AA, less likely in 3rd, due to degenerate nature = hallmark of purifying selection
- comp to human mtDNA seq data and saw conserved in humans
What did a paper looking at homoplasmic state in mice find - main findings?
(Sharpley)
- homoplasmic state gen beneficial for the mouse, therefore may have evolved to be pref homoplasmic
What did a paper looking at homoplasmic state in mice find - main findings?
(Sharpley)
- looked at 2 pops of mtDNA: NZB and 129 → prop of NZB pref reduced and 129 pref amp during oogenesis
- detailed analysis of mtDNA pops → higher levels of maternal heteroplasmy resulted in increased chance of NZB mtDNA reduction
- demonstrated selection of 1 mtDNA variant over another in female germline
- observed diff between hetero and homplasmic mice → heteroplasmic mice had reduced activity and cognitive impairment, comp to both homoplasmic strains
