ext.nuc. inheritance - basics and mt-disease

Question 1

give a brief overview of chloroplast features

Answer 1

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

Question 2

chloroplast DNA - cpDNA - give the details of it’s structure, what it encodes etc…

Answer 2

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

Question 3

what kind of numbers are we looking at for cpDNA?

Answer 3

up to 50 chloroplasts per cell, from 10 to 10,000 copies of cpDNA per chloroplast

Question 4

in the chloroplast, where is cpDNA?

what happens in cell division?

Answer 4

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

Question 5

what did Carl Correns study, and find?

Answer 5

he studied variegation (plants having white and green sections)

he found the inheritance of the property to be maternal, not mendelian

Question 6

in his experiments, what le Correns to his conclusion that variegation was maternally inherited?

Answer 6

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…

Question 7

in Correns experiment’s, what was seen when the egg came from a variegated plant, and why was this the case?

Answer 7

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

Question 8

what aspect of division would result in variegated progeny vs white vs green, from a variegated egg?

Answer 8

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

Question 9

conclusions from Corren’s experiments, and on cp-DNA

Answer 9

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

Question 10

give a brief overview of mitochondrial structure?

Answer 10

Cristae = folds of the inner membrane, inc SA to allow for more OXPHOS proteins

DNA, enzymes, ribosomes all contained in the matrix

Question 11

explain mitochondrial DNA structure briefly (shape, size, what it contains etc…)

Answer 11

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)

Question 12

what does mt-DNA encode?

Answer 12

22 tRNAs, 2 rRNAs, 13 Pps. all enzymes involved in respiration are nuclear encoded?

Question 13

what are mt-DNA’s non-coding regions?

Answer 13

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

Question 14

what numbers are we looking at for mt-DNA?

Answer 14

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

Question 15

point on appearance of mitochondria that supports endosymbiosis?

Answer 15

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

Question 16

mt-DNA doesn’t have recombination of parental alleles like the nuclear genome. how do we get variation?

Answer 16

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

Question 17

what is RFLP and how is it used/shows mt-DNA is maternally inherited?

evidence - find paper??

Answer 17

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

Question 18

how can a mutation be introduced into mt-DNA (long-term/stable)?

Answer 18

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

Question 19

what is the out of Africa theory?

Answer 19

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

Question 20

what is a haplogroup?

why does mtDNA in African populations being more diverse than other populations indicate life began in Africa?

Answer 20

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)

Question 21

what makes mt-DNA a good tool in terms of its properties? Give specific examples of how it has been used

Answer 21

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

Question 22

what are the 13 peptides the mt-genome encodes?

Answer 22

these are subunits for the different complexes in the electron transport chain

Question 23

what are the five complexes in the electron transport chain?

Answer 23

Complex I = NADH dehydrogenase

Complex II - ALL NUCLEAR ENCODED = succinate dehydrogenase

Complex III = cyt. C reductase

Complex IV = cyt. C oxidase

ATP synthase

Question 24

which of the ETC complexes is fully encoded by the nuclear genome?

Answer 24

complex II = succinate dehydrogenase

Question 25

aside from complex II, what else does the nuclear genome encode and what is the importance of this?

Answer 25

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

Question 26

explain how mtDNA replication is dependent on transcription

Answer 26

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

Question 27

mtDNA has ___ origins of replication?
most genes are encoded by the ___?

Answer 27

two origins of replication, OH and OL

most genes are encoded by the heavy strand (few by the light)

Question 28

POLG?

Answer 28

the only mtDNA polymerase, and it’s nuclear encoded

Question 29

Larsson et al. 1998 - TFAM knockout mice experiment?

Answer 29

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)

Question 30

how do levels of TFAM (potentially) directly control copy number?

Answer 30

TFAM required for transcription of LSP and HSP - which is required for mtDNA replication → so TFAM required for DNA replication

Question 31

how is mtDNA packaged?

what is the functional importance of this packaging?

Answer 31

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

Question 32

Ngo et al., 2011looked at the structure of TFAM, what did they discover about how it works?

Answer 32

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

Question 33

(kind of already said but) how do the mtDNA and nucleus work together?

Answer 33

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

Question 34

what is cross-species compatability?

Answer 34

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

Question 35

in terms of mitochondrial disease, is homoplasmy the ‘norm’/healthy?

Answer 35

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

Question 36

considering research shows low levels of heteroplasmy in healthy individuals, what are these mutations and how common are they?

Answer 36

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…

Question 37

what is meant by threshold level?

how common is mitochondrial disease?

Answer 37

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)

Question 38

don’t forget about the term mitochondrial disease…

Answer 38

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)

Question 39

broadly speaking, what kind of diseases are seen when mitochondrial function is the cause?

Answer 39

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…

Question 40

what three things determine if mt-DNA mutations will cause mitochondrial disease?

Answer 40

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

Question 41

is mitochondrial disease guaranteed to be passed on from mother to every child? explain your answer

Answer 41

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…)

Question 42

LHON (Leber’s hereditary optic neuropathy) - what is it, what causes it, prevalence?

Answer 42

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

Question 43

MERRF - myoclonic epilepsy and ragged red fibre disease

symptoms?
mutations?
why is there such a huge variation in severity between different patients?

Answer 43

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

Question 44

KSS - Kearns Sayre Syndrome

symtpoms?
what mutations?
what is the threshold like?

Answer 44

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

Question 45

what happens when POLG is mutated?

Answer 45

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

Question 46

Zhu et al., 2014 - looked at different pedigrees to investigate inheritance of mt-disease.

they saw?

they concluded?

Answer 46

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

Question 47

in what other diseases are mt-DNA mutations implicated?

Answer 47

Specific mutation associated with diabetes
Specific mutation associated with chronic fatigue
Some with cancer
Ageing - more ROS, more damage to cells and tissue