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Genetics of cell growth and division > Jones 1-4 > Flashcards

Jones 1-4 Flashcards

1
Q

What is the mtDNA structure?

A

Ds closed circle
No introns
16.6kb

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2
Q

What does mtDNA encode?

A

13 Polypeptides
22 tRNAs
2rRnas

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3
Q

What are the non-coding regions of mtDNA?

A 
D-loop: displacement loop
HSP
LSP
OH
OL
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4
Q

What is the mitochondrial structure?

A

Double membrane
Cristae
Matrix:various proteins and nucleoids

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5
Q

What does mitochondrial transcription and replication depend on?

A

Intergenomic communication

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6
Q

What are the feature of the mitochondrial genome?

A 
Vary in size 
Vary in presence of introns
Variable copy number (20 - 200,000)
Multiple genomes per organelle
Ribosomes differ
Variation in triplet code
Bacterial origin
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7
Q

How is the electron transport chain formed?

A

Assembly of multiple polypeptides
Subunits from both genomes (13/90 mitochondrial)
Complex 2 is nuclear encoded
Genomes must effectively communicate and interact

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8
Q

Key features of MtDNA replication

A 
Two origins of replication
most genes are on H strand
some on light
Transcription starts from HSP and LSP
TFs, co-factors, polymerases are nuclear encoded and depend on nuclear genome
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9
Q

Process of mtDNA replication

A
  1. TFAM binds LSP
  2. Complexes with other TFs
  3. Transcription of L strand generates a short primer for replication
  4. Transition from RNA to DNA on CSBs
  5. PolG is recruited
  6. Replication of H strand from HSP
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10
Q

What is PolG?

A

A mitochondrial DNA polymerase

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11
Q

What is TFAM?

A

Mitochondrial transcription factor A

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12
Q

Features of TFAM

A

Essential for mtDNA replication
Levels of TFAM may directly control mtDNA copy number
TFAM has an important packaging role and is the most abundant protein in mitochondrial nucleoids.
TFAM 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.

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13
Q

What do nucleoid proteins do?

A

Regulate the stability, replication, transcription and segregation of mtDNA

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14
Q

What did the Larson et al., 1998 study do?

A

Homo and heterozygous knockouts

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15
Q

What did the Larson et al., 1998 study conclude about heterozgotes?

A

Reduced mtDNA copy number
Reduced mtDNA transcription
ETC dysfunction

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16
Q

What did the Larson et al., 1998 study conclude about homozygotes?

A 
No TFAM produced
Severe mtDNA deletion
OXPHOS abolished
Enlarged mitochondria
Growth retardation
Early death - embryonic day E10.5
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17
Q

What did the Larson et al., 1998 study conclude?

A

TFAM essential for maintaining mtDNA copy umber and ETC function

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18
Q

How is there interplay between the nuclear genome and mtDNA?

A

Recognition site changes will affect the binding of transcription factors
Nuclear-encoded enzymes are required for mtDNA transcription and replication
Co-assembly of nuclear-encoded and mtDNA-encoded subunits of respiratory chain complexes
Majority of proteins that function in the mitochondria are nuclear encoded
Over 1000 nuclear-encoded products are essential to mitochondrial function
Some level of cross-species compatibility

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19
Q

How is mtDNA inherited in humans?

A

Uniparentally/maternally

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20
Q

How is mtDNA inherited in yeast?

A

Biparentally

get a heteroplasmic population that reverts to homoplasmy in 20 cycles

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21
Q

Why can yeast survive the loss of mitochondria?

A

They are faculative anaerobes

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22
Q

Why is there variation in mtDNA?

A

No parental recombination
Faster mutation rate (X10)
high variation in D loop

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23
Q

Why is the mutation rate faster in mtDNA?

A

No protective histones
lack of proofreading by PolG
high conc of free radicals generated by ETC
decreased level of repair

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24
Q

How does heteroplasmy arise?

A

age-related mutations
inheritance of a germ line mutation
introduction of foreign mitochondria to reconstructed embryos

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25
What is the mt botleneck?
Parent population has a drastic reduction in population Population of surviving individual is different The next population is different
26
Theories for mt bottleneck? (3)
a) Passive reduction of mtDNA Random segregation during cell division Reduction in copy number through divisions b) Packaging into homoplasmic clusters Segregation of nucleoids or groups of nucleoids c) Focal replication of mtDNA Selective amplification of certain mtDNA molecules
27
Stewaret et al., 2008
Organism: Mouse Purifying selection against non-synonymous mutation in proteins coding for genes of mtDNA there is a mechanism for functional testing to prevent transmission of mutated genome
28
What were Wai's conclusion
Selective amplification of certain mtDNAs occurs during ooycyte maturation Heteroplasmy levels can change dramatically between generations. Less mtDNA in primordial germ cells than ooycytes but unclear how much less Lack of mtDNA replication during early embryogenesis Selection of beneficial mtDNA variants over mutants
29
which theory of the mt bottleneck is correct?
No conclusion on which of these theories is correct
30
How is paternal mtDNA removed
Dilution effect mtDNA copy number reduced during spermatogenesis in various organisms Active degradation in fertilised ooycte
31
Nishimura et al,. 2006
Organism: Japenese meduka Elimination of sperm mtDNA upon fertilisation 1. Gradual decrease on mitochondrial nucleoids during spermatogenesis 2. Rapid digestion of sperm upon fertilisation mtDNA digestion before destruction of mitochondrial structures
32
De luca and O'Farrell, 2012
Organism: Drosophila melongaster mtDNA is removed from sperm mitochondrion prior to fertilisation 1. Endonuclease G degrade nucleoids in sperm - removes mtDNA from mitochondria 2. Back up mechanism - Actin containing investment cone sweeps all nucleoids to the apical end into a waste bag
33
What happens to the level of mtDNA in the mature sperm compared to the spermatogonia?
they are reduced
34
What happens to sperm TFAM levels through development?
They are reduced
35
What happens to TFAM levels through puberty?
Decreases
36
Where is the TFAM levels lower?
The testis
37
Sato 2011
Organism: C.Elegans Autophagy is involved in degrading serm mitochondria immediately after fertilisation sperm components activate autophagy
38
Rojansky et al., 2016
Paternal mitochondria is degraded by mitophagy Requires OXPHOS segregation of paternal mitochondria is key for degradation MUL1 and PARKIN work redundantly to degrade pathernal mitochondria
39
Sato and Sato 2017 | C.Elegans
Degredation by autophagy autophagosome encloses paternal mitochondria autophagosome fuses with lysosome degrated by ubiquiting-proteosome pathway dependent on CPS-6
40
Sato and Sato 2017 | Drosophila
mainly eliminated in spermatogenesis paternal mitochondria fuse elongate and form 2 very long mitochondria nucleoids gradually disappear Endo G responsible for initial degradation degradation by endocytic and autophagic systems
41
Sato and Sato 2017 | Vertebrates
2 step digestion before and after fertiliastion
42
Sato and Sato 2017 | Chinease hamster
Ubiquitination of MIM proteins may be involved
43
Sato and Sato 2017 | Mechanisms of active destruction
1) degradation of paternal mtDNA before and after fertilisation 2) Blocking paternal mtDNA from entering ooycte 3) elimination by autophagy and/or ubiquitination 4) uneven distrubution of paternal mitochondria
44
Sharply at al., 2012
Generated heteroplasmic mice Showed selection of one mtDNA varient over the other Showed heteroplasmic mtDNA negatively affects phenotype
45
What is the desired mitochondrial state?
Homoplasmy
46
When does heteroplasy increase?
As we age
47
Are all mtDNA changes disease associated?
No
48
How common is mitochondrial disease?
1 in 200
49
What is mitochondrial disease?
any disease that results from impaired mitochondrial function – this can be both nuclear and mtDNA mutations
50
What level of heteroplamy do you need for mitochondrial disease?
threshold level
51
What does mitotic segregation lead to?
Conversion of heteroplasmy to homoplasmy offspring with different mutant loads can get selection against negative mutations
52
Which tissues have a higher OXPHOS requirement?
Muscles, heart and neurons
53
where is mitochondrial disease most evident?
In energy extensive tissues
54
What results in mitochondrial disease?
mtDNA nuclear genes encoding OXPHOS subunits proteins required for their translation and assembly
55
How are mitochondrial disorders in nuclear genes inherited?
Mendelian inheritance
56
How are mitochondrial disorders in mt genes inherited?
Maternal inheritance | follows laws of population dynamics
57
Points to consider in inheritance of mitochondrial disease
o Random segregation during oogenesis and embryogenesis – variable mutant load o Threshold levels of mutant load o Tissue specific OXPHOS requirements (same threshold in different tissues may/may not result in disease) o Random segregation leading to variable mutant load in tissue
58
Zhu et al., 2014
Studies pedigrees with aminoglycoside-induced and non-syndromic hearing impairment looked at age of onset, severity and heteroplasmy level
59
Zhu et al., 2014 conclusions
Risk for deafness increased with increasing mutant load but the level of heteroplasmy did not directly correlate with disease severity Therefore mutant load is a very important determinant of phenotype but it is not the only variable
60
What is PolG?
Polymerase gamma on chr 15q25 22 coding exons
61
What changes can occur to PolG?
* Dominant mutations * recessive mutations * single nucleotide polymorphisms * exon 2 CAG repeat * Intronic variants
62
how many POLG mutations are missense?
94%
63
What doe PolG mutations result in ?
secondary mutations in mtDNA
64
What disease has mtDNA mutation been associated in?
Ageing, cancer diabetes and neurological disease
65
Why do we use yeast as a model?
* 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 (facultative anaerobes) * Growth phenotype simple to assess * Yeast become homoplasmic within a few generations
66
how can infertility be treated?
Cytoplasmic transfer patient eggs don't have enough mtDNA extra ooyplasm increases number of mt genomes
67
new legislation
* Approved by the Human Fertilisation and Embryology Authority on December 15, 2016 * UK has become first country to allow mitochondrial ‘replacement’ * Allow women carrying mitochondrial disease to have babies without disease * PNT or spindle transfer
68
How does spindle cell transfer occur?
1. Unfertilised patients egg with abnormal mitochondria and unfertilised donated egg with normal mitochondria – eggs are arrested at metaphase 2 2. Spindle and associated chromosomes removed as karyoplast from patient’s egg and fused into “enucleated” donor egg – spindle and associated chromosomes removed as karyoplast from donated egg and discarded 3. Reconstructed egg is fertilised (by ICSI) with sperm from patient’s partner 4. Cleaving embryo with normal mitochondria and maternal and paternal genome can be transferred to the uterus
69
How does pronuclear transfer occur?
1. Patients egg with abnormal mitochondria fertilised with partners sperm. The donated egg is also fertilised – normal mitochondria 2. Patients zygote with abnormal mitochondria and zygote – normal mitochondria 3. Patients pronuclei removed from zygote and transferred to enucleated egg, which has normal mitochondria 4. Leaving embryo with normal mitochondria and maternal and paternal genome can be transferred to the uterus
70
How can you get mitochondrialdisease after PNT or SCT?
level of heteroplasmy in embryo | random segregation can convert that to homoplasmy
71
Somatic cell nuclear transfer
• Reported contributions of donor mtDNA: o 0-63% in embryos o 0-59% in offspring o Have preferential replication of donor DNA • Donor mtDNA absent in some intra-specific crosses but not all • Donor mtDNA detected at variable levels in inter-specific or cross-species crosses • Persistence is unpredictable • Overcome by mtDNA depletion of donor cells
72
How were mitochondria created?
Symbiosis event
73
Functions of mitochondria
Cellular energy metabolism biogenesis of Fe-S clusters Apoptotic cell death
74
How does mtDNA mutation contribute to cancer?
Contributes to ooygenesis/metastasis
75
What does a low level of heteroplasmy inetracting with de novo somatic mutation cause?
Exacerbate ageing and neurological phenotypes
76
What happens in no-dividing cells?
Mt DNA is continuously made and destroyed some replicated more frequently changes level of heteroplamy
77
how does mtDNA vary in the population?
Some varients have remained restricted to certain ethnic groups
78
what is the point mutation in Diabetes and deafness
3243 on mtDNA
79
What gene is affected in diabetes and deafness?
gene encoding tRNEleu
80
how is diabetes and deafness inerited?
maternally
81
how is diabetes and deafness caused?
Decreased OXPHOS decreased ATP affect insulin production may affect ion umps required for sound transmission
82
What does Leigh syndrome affect?
heart, muscles, eyes. lungs, neurological system
83
How is Leigh syndrome caused?
mutations in mtDNA or nuclear genome affects 4/5 OXPHOS complexes disordered OXPHOS
84
what mtDNA mutation causes leigh syndrome?
Mutation in ATPsynthase subunit
85
What nuclear mutation causes leigh syndrome?
Many e.g. COX

Genetics of cell growth and division (4 decks)

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