Lecture 24 - Extranuclear inheritance III Flashcards
(24 cards)
What is neurospora crassa?
A bread mold - slow growing mutant strain, named poky, has impaired mitochondrial function due to mtDNA mutations
- Neurospora has 2 mating types - designated A & a which fuse together; both A & a can be maternal depending on fertilization act.
- Performed reciprocal crosses between wild-type & poky strains, varying whether maternal parent was WT or poky
What was the result of reciprocal crosses between WT & poky strains, varying whether maternal parent was WT or poky.
Maternal parent (Poky) crossed with phenotypically normal paternal source - shows nuclear mutation.
Offspring spores - show mendelian inheritance of nuclear gene, but phenotype resulting from mitochondrial dysfunction. HOWEVER - when Poky maternal - all offspring spores has Poky mutation. Same is true when normal is maternal (then offspring normal).
Offspring were either all poky or all wild-type depending on which parent had poky genotype: this revealed maternal inheritance of poky phenotype & hence of mtDNA mutations.
Mutations in cytoplasmic organelles are inherited maternally & separately to the Mendelian inheritance patterns of nuclear genetic mutations.
What are Petites in Saccharomyces cerevisiae?
- Petites (small colonies): result from deficiency in cellular respiration due to DEFECTIVE ELECTRON TRANSPORT CHAIN
- Facultative anaerobe: can generate energy through glycolysis; survives the loss of mitochondria function.
What are the 3 different types of petites?
- Segregation petites
- Neutral petites
- Suppressive petites
What are segregational petites?
Crossing haploid petite with haploid normal generates diploid zygotes & offspring spores was 50:50 petite:normal (mendelian inheritance due to nuclear mutations)
What are neutral petites?
crossing haploid petite with haploid normal generated 100% normal. Initially get combining of mitochondrial network of both parent cells & mixing of mtDNA. After 20 cell divisions, one population of mtDNA has been selected. Example of initially mtDNA inheritance is bi-parental, but not sustained.
What are suppressive petites?
Crossing haploid petite with haploid normal generated all petite. Not completely understood, but initially mixing hasn’t resulted in maintenance of normal mtDNA. Could be that genome has recombined & mutated or preferential replication, so it is always maintained.
How can we use yeast models to study mitochondrial disease?
- mitochondrial functions are highly conserved between humans & 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 of non-fermentable carbon sources such as glycerol, ethanol or lactate.
Describe growth dynamics under respiratory conditions for WU12 & DU23 interspecific hybrids.
WU12 - cross of the W1 S. cerevisiae strain with the U2 S. uvarum strain
DU23: cross of the D2 S. cerevisiae strain with the U3 S. uvarum strain
Some are growing quicker, showing that selection of mtDNA does impact oxidative phoshorylation capacity, and in turn function of the mitochondria.
What is an experiment where yeast models to study mitochondrial disease?
Influence of 4 different nuclear contexts on the growth phenotype of the LeuA30(29)G mutation. Equivalent to human mitochondrial tRNA mutations that results in MELAS (mitochondrial disease)
Yeast can only grow if oxidative phosphorylation is occurring. Looking at 4 different nuclear contexts, with same mtDNA mutation. Highlights mitochondrial disease is impacted by secondary mutations/polymorphisms within the nuclear genome.
Growth is different - same mitochondrial mutations, just different nuclear contexts. This shows nuclear DNA is a contributor to variable disease severity so often observed with mitochondrial disease.
What are Parental mtDNA contributions in mammals?
Oocyte - 150k - 200k copies mtDNA
Sperm - 10 copies mtDNA
Destruction of paternal mtDNA following fertilization
Cell type with highest copy number is mature oocyte
Elegant mechanisms to actively destroy paternal mtDNA, following fertilization.
How does reduction of paternal mtDNA occur?
- dilution effect due to higher copy numbers in oocytes than in sperm
- mtDNA copy number reduced during spermatogenesis in various organisms
- Reduction of mtDNA & cpDNA during pollen development in Arabidopsis.
- Active destruction of mtDNA & cpDNA.
The levels of mtDNA are much reduced in the mature sperm than in the spermatogonia. There is also a reduction of sperm TFAM levels throughout development.
What was found regarding spermatogenesis in rat testes?
During spermatogenesis, immature sperm reside on the outside, near the basement membrane, and travel inwards the lumen of the tubule during maturation.
Brighter areas on the outside, where mtDNA is. This backs up idea that mtDNA reduces in copy number as sperm mature.
COPY NUMBER CORRELATES WITH TFAM LEVELS.
Western blot showing TFAM levels in different organs. TFAM levels are low in testes.
Decline in TFAM levels in testis through puberty.
How does elimination of paternal mtDNA occur?
Active degradation of paternal mtDNA following fertilization in many species
- role of ubiquitin in mammals including cows & humans
- Paternal mtDNA destroyed by autophagy in C. elegans.
This prevents sperm entering into the egg.
Paternal mtDNA tagged with ubiquitin, which differed from other mtDNA. The ubiquitin tag are tagging themselves for degradation.
Leakage of paternal mtDNA isn’t the norm - but can occur. Can also lead to disease
What are sources of heteroplasmy?
- age-related mutations
- inheritance of a germline mutation
- paternal mtDNA leakage
- introduction of foreign mitochondria to reconstructed embryos
What is homoplasmy, heteroplasmy & mtDNA bottleneck?
Heteroplasmy arises in the body as we age. Homoplasmic population is needed. In order to encourage homoplasmy (or prevent Heteroplasmy) - the bottleneck concept is introduced. The bottleneck describes a dramatic reduction in mtDNA copy number (which follows fertilisation).
There is a sampling of the population & the surviving individuals are the ones passed on. They will then be amplified, giving rise to the mtDNA population for the next generation.
Bottleneck effect is coming into play when making oocytes for the next generation.
What are different theories for the mtDNA bottleneck?
passive reduction of mtDNA - random segregation during cell division
packaging into homoplasmic clusters (nucleoids) - segregation of nucleoids or groups of nucleoids
focal replication of mtDNA - selective amplification of certain mtDNA molecules
Describe features of the mtDNA bottleneck
Fertilized oocyte - high mtDNA copy number. Following fertilization, oocyte doesn’t grow in size, but zygote undergoes cleavage divisions, with the cells getting smaller. During early embryo development, there is not active replication of mtDNA - every cell has a smaller & smaller copy number - this means there is a huge difference in mitochondrial genome population size between oocyte & primordial germ cells.
Primordial germ cells are only one set of cells in the oocyte, but will become oocytes in the next generation. During embryo development the mitochondrial population of baby’s children are determined.
After primordial germ cells, there is development of oogonia - going to become oocytes. They develop up to the primordial follicle cells (during foetal development), then arrest part way through meiosis, then following puberty, certain oocytes are recruited each month to mature into mature oocytes, which is larger in size. With increase in size, very active replication of mtDNA, such that the copy number is increasing.
What are the 2 points at which the bottleneck is acting?
- Between fertilized oocyte to primordial follicle
- primary oocyte to mature oocyte.
Oocyte development occurs when TFAM expression & replicating mtDNA overlap.
Selective amplification of certain mtDNAs occurs during oocyte maturation.
This means that heteroplasmy levels can change dramatically between generations
Certain genomes can be selected over others & dramatic shifts in populations between generations.
What model of the mtDNA bottleneck is correct?
mtDNA copy number declines in PGCs (primordial germ cells)
- likely to be a combination
- plenty of evidence to show lack of mtDNA replication during early embryogenesis
- Multiple sources of experimental evidence that show POSITIVE SELECTION of beneficial mtDNA variants over mutants & also PURIFYING SELECTION to remove deleterious mutants
- points of concentration around copy number. Some controversy
What are assisted reproductive technologies?
UK has become first country to allow mitochondrial ‘replacement’. Newcastle Fertility clinic was granted the first licence.
Allows women carrying mitochondrial disease to have babies without disease
Pronucleus transfer (PNT) or spindle transfer.
A number of different techniques used - including Pronuclear transfer (PNT) or spindle transfer.
What are the stages of Spindle transfer?
- Unfertilized patient’s egg with abnormal mitochondria.
- Spindle & associated chromosomes removed as karyoplast from patient’s egg & fused into ‘enucleated’ donor egg.
- Reconstituted egg is fertilized by (ICSI - Intracytoplasmic Sperm Injection) with sperm form patient’s partner
- Cleaving embryo with normal mitochondria & maternal & paternal genome can be transferred to the uterus.
In this case, all of mtDNA will come from donor egg, and nuclear material is coming from parent.
What are the stages of Pro-nuclear transfer?
- Patient’s egg with abnormal mitochondria fertilized with partner’s sperm.
- Patient’s zygote with abnormal mitochondria
- Patients’ pronuclei removed from zygote & transferred to enucleated egg.
- Cleaving embryo with normal mitochondria & maternal and paternal genome can be transferred to the uterus.
Both patient & donor egg are fertilized. Develop till pronuclear stage - male & female pronuclei come together at the centre of the fertilized oocyte. The pronuclei are taken out of donor egg & pronuclei from patient & part er inserted instead.
What happens to the mutants mtDNA (if transferred to oocyte)?
Can happen due to mitochondrial cluster around the metaphase 2 spindle. It is often the case in reconstructed oocytes that some donor mtDNA will be transferred.
