mt-DNA in other organisms, paternal outcome, fertility Flashcards
what is neurospora Crassa?
train of mould, where some was seen to be slower at growing, identified to be a result of an mtDNA mutation, called poky
This mould as A and a mating types, either of which can be maternal
what experiment was done in neurospora Crassa
Communicated by G. W. Beadle, March 3, 1952
Investigated inheritance of the mt-mutation poky by performing reciprocal crosses, comparing offspring from maternal WT + paternal poky, vs maternal poky and paternal WT
Also added a nuclear mutation for comparison
Results -
Mendelian inheritance of nuclear mutant, 50/50 in both scenarios
Offspring all matched maternal phenotype, suggesting maternal inheritance
what are petites in yeast?
mtDNA mutations/mutations affecting mitochondrial function identified as ‘petites’ (small colonies due to insufficient respiration - defective e- transport chain)
explain what is meant by segregational vs neutral vs supressive petites?
when crossing a haploid ‘petite’ and a normal haploid in yeast, three outcomes were observed -
segregational -
50/50 petites vs WT, i.e. showed mendelian inheritance, meaning petites can arise from nuclear mutations (we know this idea)
neutral - all normal progeny regardless of which parent cell was mutant -
In yeast inheritance of mtDNA isn’t uniparental, the yeast fuse, the diploid has both mutant and WT genomes for about 20 divisions after which one is selected/reverts to homoplasmy (and in this case WT was selected)
suppressive -
all progeny were petites, meaning mutations can behave dominantly. yeast selected the mutant mt-DNA when reverting to homoplasmy. could be preferentially replicated e.g. the mutant genome is shorter so quicker to replicate
why are yeast useful for mitochondrial disease research?
Mitochondrial functions are highly conserved between humans and Saccharomyces cerevisiae
Possible to undertake large scale screens; genetic manipulations are easy; biochemical analyses well established
Yeast can survive on fermentable carbon sources in the absence of mitochondrial function
Growth phenotype simple to assess; use a non-fermentable carbon source e.g. glycerol/ethanol/lactate (only ones with function ETC can survive)
Crossed two different strains of yeast, S. cerevisiae and S. uvarum, what did they look at and conclude?
Compared the ones that selected Su mt-DNA (blues) when reverting to homoplasmy, vs Sc mt-DNA
So they were looking at yeast with the same nuclear background (all had a mix of the two nuclear genomes) but two different mt-genomes (because one or the other is selected)
Conclusions -
Provided evidence for the idea that the ‘nuclear context’ is important - i.e. the compatibility between the nuclear products in the mitochondria and the mt-genome products. You can see the Su mt-DNA yeast grew a bit slower in both experiments
De Luca et al., 2009
showed the importance of nuclear context, how?
Influence of four different nuclear contexts (idk different alleles for genes involved in mt-function) on the growth phenotype of the mt-DNA LeuA30(29)G mutation (equivalent to human mitochondrial tRNA mutations that result in MELAS)
using serial dilutions for easier viewing, saw differences in growth for the same mt-DNA mutation in the different nuclear contexts
Concluded different nuclear contexts resulted in differences in growth for the same mt-DNA mutation
***Therefore - Secondary nuclear mutations can have an impact on phenotype
name the three things that happen to paternal mtDNA (explain the easy one)
dilution - Oocyte: 150,000-200,000 copies mtDNA
Sperm: as little as ~10 copies mtDNA
Reduction of copy number in sperm maturation
Destruction of paternal mt-DNA post fertilisation
give examples/evidence/explanation of the idea of reduction in mt-DNA copy number in sperm maturation
Rantanen et al., 2001
Sperm production occurs in seminiferous tubules, with immature sperm at the edge and mature moving into the centre
Visualisation of mtDNA and RNA and TFAM showed much more at edges - in immature sperm/spermatogonia - than centre in mature sperm, indicating a decline in mtDNA copy number
Also - western blot for TFAM across the body (in rats) showed testes lower levels than the heart, liver, kidneys
AND that TFAM levels declined with age - through puberty
give examples/evidence/explanation destruction of paternal mt-DNA post fertilisation
what was an interesting observation across all the experiments?
Ubiquitin tags on paternal mt-DNA followed by degradation has been shown in cows and humans (Sutovsky et al., 1999)
Paternal mtDNA destroyed by autophagy in C. elegans (Sato and Sato, 2011; Zhou et al., 2016), Drosophila (Politi et al., 2014) and mouse (Rojansky et al., 2016)
^^ the mechanisms in the different species are all slightly different, indicating the concept of destroying paternal mt-DNA is important
give four sources of heteroplasmy
age-related mutations
inheritance of a germline mutation
paternal mtDNA leakage (doesn’t get destroyed)
introduction of foreign mitochondria to reconstructed embryos
with all those sources of heteroplasmy, there must be mechanisms to reduce it/control it.
what are the three main ideas here?
Passive reduction -
The most basic idea, basically that there isn’t any ‘mechanism’ per say, but that the massive reduction in mt-DNA between the parent as a whole, to the oocyte, + random segregation acting during the process of those oocyte’s production
Segregation of nucleoids -
The idea that the unit in which the mt-genomes are separated are in clusters of homoplasmic nucleoids
Focal replication of mt-DNA -
Some people think it’s not enough to explain the observed differences between offspring and mother, and that some mt-genomes must be amplified/selected
what is the mt-bottleneck?
The fertilised oocyte undergoes loads of divisions, making loads of cells, some of which are primordial germ cells (females born with all their eggs etc…)
This transition has a huge reduction in cell size and therefore mtDNA number, ***especially considering mt-DNA is not replicated for the earlier stages of embryogenesis
(then huge increase in copy number)
From puberty, the oocytes continue maturation; this involves a huge increase in size and an amplification of mt-DNA copy number. This is where the preferential replication idea would occur, dictating the mt-DNA makeup of the oocyte and offspring
Wai et al., 2008 on preferential replication?
The paper (Wai et al., 2008) argues we see this selective amplification of mitochondrial DNA at this point (egg maturation in puberty with the massive inc. in mt-DNA copy number) and ***therefore a big increase in heteroplasmy or gene variants
what did Wai et al., 2008 paper show/do and what did they conclude? (selective amplification etc…)
visualised newly replicated mt-DNA in green
TFAM in red
Overlay and if they match up perfectly you get yellow = where both TFAM and replicated mtDNA are
The yellow dots are a subset of the red dots, so only some of the mtDNA present are being actively replicated
Wai’s Conclusion:
Selective amplification of certain mtDNAs occurs during oocyte maturation.
This means that heteroplasmy levels can change dramatically between generations
