ext.nuc. inheritance - basics and mt-disease Flashcards
give a brief overview of chloroplast features
Present in green plants, photosynthetic protists, and blue-green algae; site of photosynthesis
Outer membrane and inner membrane
Stroma: fluid which contains DNA and ribosomes
Thylakoids: membrane-bound structures containing chlorophyll, stacks called grana
Photosystem proteins found in thylakoid membranes
chloroplast DNA - cpDNA - give the details of it’s structure, what it encodes etc…
Double stranded and circular, commonly 100-225 kb long
Does contain introns
Low GC content, around 36%
Genes are arranged in operons like in prokaryotes
Has two inverted repeats, varying in length, 4 - 150 genes long (10-76 kb)
Encodes - subunits of photosynthetic machinery (some are nuclear encoded), Ribosomal proteins, polymerases, tRNAs and rRNAs
what kind of numbers are we looking at for cpDNA?
up to 50 chloroplasts per cell, from 10 to 10,000 copies of cpDNA per chloroplast
in the chloroplast, where is cpDNA?
what happens in cell division?
cpDNA is associated with the thylakoid or inner membrane
chloroplasts divide by binary fission and must be inherited during cell division
cpDNA is replicated BEFORE chloroplast division
what did Carl Correns study, and find?
he studied variegation (plants having white and green sections)
he found the inheritance of the property to be maternal, not mendelian
in his experiments, what le Correns to his conclusion that variegation was maternally inherited?
when the egg was white or green, the progeny phenotype always matched (the egg/mother) despite whatever the pollen phenotype was
when the egg was from a variegated plant tho…
in Correns experiment’s, what was seen when the egg came from a variegated plant, and why was this the case?
the progeny were either, white, green or variegated
this is due to the idea of heteroplasmy and homoplasmy
heteroplasmy = when a cell contains a mixture of WT and mutant mt/cp genomes
homoplasmy = when a cell contains identical mt or cp
genomes
what aspect of division would result in variegated progeny vs white vs green, from a variegated egg?
all due to random segregation
when a cell (like a primordial germ cell) divides, mitochondria are separated entirely randomly, so its possible to get oocytes where one has ended up with all mutant mt-genomes and so that progeny is white, while the other got all the WT mt-genomes, so gives rise to green plants
most common would be the primordial germ cell being heteroplasmic (containing both mutant and WT DNA) and random segregation resulting in oocytes that are also heteroplasmic, giving variegated progeny
conclusions from Corren’s experiments, and on cp-DNA
Chloroplast DNA mutation leads to loss of chlorophyll
Chloroplast DNA is inherited from the egg only = maternal inheritance
Variegation results from wild-type and mutant tissue
Individual cells can contain a mixture of wild-type and mutant genomes = heteroplasmy
give a brief overview of mitochondrial structure?
Cristae = folds of the inner membrane, inc SA to allow for more OXPHOS proteins
DNA, enzymes, ribosomes all contained in the matrix
explain mitochondrial DNA structure briefly (shape, size, what it contains etc…)
Double stranded, closed circle
Generally smaller than cpDNA 16-18kb in mammals (but can be larger in yeast and plants)
Introns super rare (seen more in the larger sized ones like yeast)
what does mt-DNA encode?
22 tRNAs, 2 rRNAs, 13 Pps. all enzymes involved in respiration are nuclear encoded?
what are mt-DNA’s non-coding regions?
D-loop - displacement loop (or control region)
HSP - H (heavy)-strand promoter region,
LSP - L (light)-strand promoter region
OH - H-strand origin of replication and
OL - L-strand origin of replication
what numbers are we looking at for mt-DNA?
Multiple copies of mtDNA in each organelle, multiple organelles per cell, it varies
Lowest no. of copies is in sperm, 20-100 mt-DNA
Somatic cells tend to be 1000-10,000
Highest is in oocytes, over 150,000 mt-DNA
point on appearance of mitochondria that supports endosymbiosis?
mitochondrial ribosomes appear more like bacterial ones (suggesting bacterial origins, endosymbiosis)
In the nuclear genetic code, UGA is a stop codon, whereas in the mitochondrial genetic code UGA codes for tryptophan; AUA codes for isoleucine in the nuclear genetic code, whereas AUA codes for methionine in the mitochondrial
mt-DNA doesn’t have recombination of parental alleles like the nuclear genome. how do we get variation?
mtDNA has a faster mutation rate (est. 10X to 20X) than nuclear DNA; no protection from histones; poorer repair mechanisms
Particularly high level of variation in the D loop (control region) of mtDNA
what is RFLP and how is it used/shows mt-DNA is maternally inherited?
evidence - find paper??
RFLP = Restriction Fragment Length Polymorphism
Used a polymorphism resulting in absence or presence of a certain restriction site, so application of enzyme results in cutting or no cutting, one big band or two small bands in gel electrophoresis
Phenotype of offspring matches mother’s regardless of father
how can a mutation be introduced into mt-DNA (long-term/stable)?
Mutations can occur in a copy of mtDNA in oocytes, will be passed on
This may initially result in heteroplasmy
Over time, this mutated copy is replicated a bit more, passed on etc… random segregation of mitochondria can create a homoplasmic population
The polymorphism is then stably inherited by all future generations
what is the out of Africa theory?
analysis of mt-DNA, archaeological samples etc… found that mtDNA in African populations is more diverse than other populations and genetic diversity in non-Africans is a subset of that found in Africans
what is a haplogroup?
why does mtDNA in African populations being more diverse than other populations indicate life began in Africa?
Mitochondrial haplogroup can be defined as a population who share similar mtDNA sequence (or have similar mtDNA sequence changes or polymorphism)
founder effect - reduced population = reduced diversity (and the diversity that is present would be a subset of the OG-population (Africans))
populations that moved out of Africa would be the ‘newer’ groups to have formed compared to populations already in Africa, and so have had less time to generate as much diversity (especially since they started from a small subsection - founder effect again)
what makes mt-DNA a good tool in terms of its properties? Give specific examples of how it has been used
Lack of recombination; clearer inheritance patterns between generations
High cell copy number; more copies present in the cell leads to higher chance of preservation when looking at e.g. old mummified specimens to something
MtDNA has been permissible as evidence in US courts since 1996
First case involved hair samples in a murder case; it was not possible to isolate nDNA from the samples but mtDNA analysis was successful
Separate conviction of a killer as a result of cat hair mtDNA analysis
what are the 13 peptides the mt-genome encodes?
these are subunits for the different complexes in the electron transport chain
what are the five complexes in the electron transport chain?
Complex I = NADH dehydrogenase
Complex II - ALL NUCLEAR ENCODED = succinate dehydrogenase
Complex III = cyt. C reductase
Complex IV = cyt. C oxidase
ATP synthase
which of the ETC complexes is fully encoded by the nuclear genome?
complex II = succinate dehydrogenase
aside from complex II, what else does the nuclear genome encode and what is the importance of this?
the nuclear genome encodes some subunits for all the complexes
Each complex requires multiple assembly proteins, these are nuclear encoded
Transcription factors, co-factors and mtDNA polymerase are all nuclear-encoded
So neither genome controls the entire transport chain
explain how mtDNA replication is dependent on transcription
the D-loop has the transcription start site, as well as origin of replication of light and heavy strand (OH and OL)
TFAM (transcription factor A mitochondrial) binds to the transcription promotors
(in complex with other proteins) and initiates transcription with RNA pol.
An entire copy of the strand is produced - polycystic copy which is later broken down
Also from that same promoter, we get transcription of an RNA primer which is essential for replication to take place
mtDNA has ___ origins of replication?
most genes are encoded by the ___?
two origins of replication, OH and OL
most genes are encoded by the heavy strand (few by the light)
POLG?
the only mtDNA polymerase, and it’s nuclear encoded
Larsson et al. 1998 - TFAM knockout mice experiment?
Used knockout mice to show that TFAM is essential in maintaining mtDNA copy number and for respiratory chain function
–/+ showed reduced mtDNA copy number and reduced MtDNA transcription, resulting in respiratory (as in energy production) dysfunction in the heart
–/– showed severe mtDNA depletion, abolished OxPhos, and was lethal (embryos died)
how do levels of TFAM (potentially) directly control copy number?
TFAM required for transcription of LSP and HSP - which is required for mtDNA replication → so TFAM required for DNA replication
how is mtDNA packaged?
what is the functional importance of this packaging?
mtDNA not completely free and floating in cytoplasm - its organised into nucleoids where it is associated with proteins, the most abundant of which is TFAM (another example is mtSSB - mitochondrial single-stranded DNA binding protein)
NOTE - these nucleoid proteins regulate the stability, replication, transcription and segregation of mtDNA
Ngo et al., 2011looked at the structure of TFAM, what did they discover about how it works?
TFAM - has four domains (HMG-box A → linker → HMG box B → C-tail)
TFAM HMG box binding at the LSP and HSP forces the mtDNA into a U-shape
This is structurally important for the activation of transcription from these two sites
(kind of already said but) how do the mtDNA and nucleus work together?
Nucleus provides enzymes essential for mtDNA transcription and replication
Both mtDNA and nuclear provide subunits of the respiratory chain proteins
Majority of proteins that function in the mitochondria are nuclear-encoded; Over 1000 nuclear-encoded products are essential to mitochondrial function
Inter-genomic communication is essential - the nucleus and mitochondria must communicate
what is cross-species compatability?
basically binding sites of a protein are super specific, so making a hybrid cell with e.g. a mouse nucleus and a rat cytoplasm results in the nuclear proteins/enzymes etc… having lower affinity for the mtDNA products so respiratory chain/mtDNA replication/transcription are affected.
But there is some cross-species compatibility seen
in terms of mitochondrial disease, is homoplasmy the ‘norm’/healthy?
in a way yes, all wt-genomes mean there isn’t mt-disease
However research shows individuals have very low levels of heteroplasmy, and that levels increase within individuals with age; tho this has been associated with ageing effects and numerous diseases
considering research shows low levels of heteroplasmy in healthy individuals, what are these mutations and how common are they?
Most are harmless polymorphisms (tho research shows effects on athletic ability for example)
Some mutations that occur can be disease-causing mutations tho…
These are more common than might expect - 1 in 200 carrying known point mutation (as in associated with disease)
also - threshold level etc…
what is meant by threshold level?
how common is mitochondrial disease?
there is a threshold level for the ‘mutant load’, above which you ‘have the disease’.
You can have a very low level of mutations/heteroplasmy but not have the symptoms of a disease
Prevalence of disease/above threshold: 1 in 4000 children born with an inherited mitochondrial disease in the US. 1 in 5000 adults affected
the reason its 1 in 200 carrying a known disease–causing mutation is cus of threshold level)
don’t forget about the term mitochondrial disease…
includes any disease that results from impaired mitochondrial function. Show maternal inheritance IF the gene mutation is an mtDNA one…
But many mitochondrial proteins are nuclear encoded and their mutations can cause mitochondrial disease (which would show mendelian inheritance)
broadly speaking, what kind of diseases are seen when mitochondrial function is the cause?
Mitochondrial disease - main problem = impact on OXPHOS rate/function
so diseases seen are ones affecting cells with high-energy usage - muscle cells (so myopathies inc cardiomyopathies). Neurons e.g. in Parkinson’s etc…
what three things determine if mt-DNA mutations will cause mitochondrial disease?
mutant load and whether it exceeds threshold level
presence of the mutation in a specific tissue - due to random segregation in development, mutation isn’t necessarily spread evenly across the whole body, and different tissues have different OXPHOS demands, so mutant load has to be reached in the tissue in question
secondary mutations - e.g. polymorphisms in nuclear encoded mt-proteins could enhance/increase likelihood of disease phenotype
is mitochondrial disease guaranteed to be passed on from mother to every child? explain your answer
NO - its not guaranteed
due to random segregation at oogenesis -
Primordial germ cell of mother = heteroplasmic, contains some mutant mt-genomes
Produces oocytes with varying degrees of heteroplasmy - high/intermediate/low levels of mutation, does not always affect offspring
NOTE - Mitochondrial disorders caused by nuclear mutations = Mendelian inheritance patterns so also a no
summary - Mitochondrial diseases caused by mtDNA mutations are maternally inherited but follow laws of population dynamics (changes in heteroplasmy/selection/bottlenecks and founder effects etc…)
LHON (Leber’s hereditary optic neuropathy) - what is it, what causes it, prevalence?
Degeneration of the retinal ganglion cells + axons
Causes acute onset blindness in young adulthood - seen more in males
Diff mtDNA point mutations seen in NADH dehydrogenase subunits, but phenotype severity is affected by many secondary mutations - genetically heterogeneous disease (can be caused by mutations in different genes)
NOTE - this adds to difficulty of diagnosing mt disease
MERRF - myoclonic epilepsy and ragged red fibre disease
symptoms?
mutations?
why is there such a huge variation in severity between different patients?
Symptoms - myoclonus (muscle seizure/jerking), myopathy, seizures, ataxia, peripheral neuropathy, dementia
Mutation in mtDNA gene ‘MT-TK’ encoding tRNALys , 8344A>G
Mitochondria unable to make proteins required for functional respiratory chain
Reiteration - Huge variation in severity between different patients and significant variations in tissue distributions of mutant mtDNA.
BECAUSE - random segregation in development results in varying mutant load in different tissues (between individuals, between organs within an individual etc…); some will not exceed threshold levels. IT’S ABOUT WHETHER THE MUTANT LOAD EXCEEDS THRESHOLD IN THE SPECIFIC TISSUE TYPE
KSS - Kearns Sayre Syndrome
symtpoms?
what mutations?
what is the threshold like?
Symptoms - Multisystem disorder, particularly CNS and eyes
Mutation - not usually maternally inherited, so must be nuclear mutation…
Mutations affecting DNA polymerase gamma (POLG), resulting in large scale deletions in the mtDNA
Threshold - as the result of the mutation is large scale deletions in mtDNA, 100% mutant load = lethal, 20-90% mutant load = disease phenotype, <20% = asymptomatic
what happens when POLG is mutated?
POLG - loads of known mutations, it’s the only mitochondrial polymerase so there’s no redundancy. When mutated you get deletions or ‘secondary mutations’, or just depletion of the mitochondrial genome
Presentation varies between patients - don’t know what mt-genes will be affected, i.e. clinically heterogeneous
Zhu et al., 2014 - looked at different pedigrees to investigate inheritance of mt-disease.
they saw?
they concluded?
in some families mothers pass on the disease to all children, in others just some, in others none
Can also see sudden appearance of disease when it wasn’t seen in the family for a few generations previous
conclusions:
Risk for deafness increased with increasing mutant load
BUT
the level of heteroplasmy did not correlate with disease severity
Therefore
mutant load is a very important determinant of phenotype but it is not the only variable
in what other diseases are mt-DNA mutations implicated?
Specific mutation associated with diabetes
Specific mutation associated with chronic fatigue
Some with cancer
Ageing - more ROS, more damage to cells and tissue
