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

Jones 1-4 Flashcards

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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
Q

What is the mt botleneck?

A

Parent population has a drastic reduction in population
Population of surviving individual is different
The next population is different

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26
Q

Theories for mt bottleneck? (3)

A

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

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27
Q

Stewaret et al., 2008

A

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

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28
Q

What were Wai’s conclusion

A

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

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29
Q

which theory of the mt bottleneck is correct?

A

No conclusion on which of these theories is correct

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30
Q

How is paternal mtDNA removed

A

Dilution effect
mtDNA copy number reduced during spermatogenesis in various organisms
Active degradation in fertilised ooycte

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31
Q

Nishimura et al,. 2006

A

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

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32
Q

De luca and O’Farrell, 2012

A

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

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33
Q

What happens to the level of mtDNA in the mature sperm compared to the spermatogonia?

A

they are reduced

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34
Q

What happens to sperm TFAM levels through development?

A

They are reduced

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35
Q

What happens to TFAM levels through puberty?

A

Decreases

36
Q

Where is the TFAM levels lower?

A

The testis

37
Q

Sato 2011

A

Organism: C.Elegans
Autophagy is involved in degrading serm mitochondria immediately after fertilisation
sperm components activate autophagy

38
Q

Rojansky et al., 2016

A

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
Q

Sato and Sato 2017

C.Elegans

A

Degredation by autophagy
autophagosome encloses paternal mitochondria
autophagosome fuses with lysosome
degrated by ubiquiting-proteosome pathway
dependent on CPS-6

40
Q

Sato and Sato 2017

Drosophila

A

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
Q

Sato and Sato 2017

Vertebrates

A

2 step digestion before and after fertiliastion

42
Q

Sato and Sato 2017

Chinease hamster

A

Ubiquitination of MIM proteins may be involved

43
Q

Sato and Sato 2017

Mechanisms of active destruction

A

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
Q

Sharply at al., 2012

A

Generated heteroplasmic mice
Showed selection of one mtDNA varient over the other
Showed heteroplasmic mtDNA negatively affects phenotype

45
Q

What is the desired mitochondrial state?

A

Homoplasmy

46
Q

When does heteroplasy increase?

A

As we age

47
Q

Are all mtDNA changes disease associated?

A

No

48
Q

How common is mitochondrial disease?

A

1 in 200

49
Q

What is mitochondrial disease?

A

any disease that results from impaired mitochondrial function – this can be both nuclear and mtDNA mutations

50
Q

What level of heteroplamy do you need for mitochondrial disease?

A

threshold level

51
Q

What does mitotic segregation lead to?

A

Conversion of heteroplasmy to homoplasmy
offspring with different mutant loads
can get selection against negative mutations

52
Q

Which tissues have a higher OXPHOS requirement?

A

Muscles, heart and neurons

53
Q

where is mitochondrial disease most evident?

A

In energy extensive tissues

54
Q

What results in mitochondrial disease?

A

mtDNA
nuclear genes encoding OXPHOS subunits
proteins required for their translation and assembly

55
Q

How are mitochondrial disorders in nuclear genes inherited?

A

Mendelian inheritance

56
Q

How are mitochondrial disorders in mt genes inherited?

A

Maternal inheritance

follows laws of population dynamics

57
Q

Points to consider in inheritance of mitochondrial disease

A

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
Q

Zhu et al., 2014

A

Studies pedigrees with aminoglycoside-induced and non-syndromic hearing impairment
looked at age of onset, severity and heteroplasmy level

59
Q

Zhu et al., 2014 conclusions

A

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
Q

What is PolG?

A

Polymerase gamma
on chr 15q25
22 coding exons

61
Q

What changes can occur to PolG?

A
  • Dominant mutations
  • recessive mutations
  • single nucleotide polymorphisms
  • exon 2 CAG repeat
  • Intronic variants
62
Q

how many POLG mutations are missense?

A

94%

63
Q

What doe PolG mutations result in ?

A

secondary mutations in mtDNA

64
Q

What disease has mtDNA mutation been associated in?

A

Ageing, cancer diabetes and neurological disease

65
Q

Why do we use yeast as a model?

A
  • 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
Q

how can infertility be treated?

A

Cytoplasmic transfer
patient eggs don’t have enough mtDNA
extra ooyplasm increases number of mt genomes

67
Q

new legislation

A
  • 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
Q

How does spindle cell transfer occur?

A
  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
Q

How does pronuclear transfer occur?

A
  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
Q

How can you get mitochondrialdisease after PNT or SCT?

A

level of heteroplasmy in embryo

random segregation can convert that to homoplasmy

71
Q

Somatic cell nuclear transfer

A

• 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
Q

How were mitochondria created?

A

Symbiosis event

73
Q

Functions of mitochondria

A

Cellular energy metabolism
biogenesis of Fe-S clusters
Apoptotic cell death

74
Q

How does mtDNA mutation contribute to cancer?

A

Contributes to ooygenesis/metastasis

75
Q

What does a low level of heteroplasmy inetracting with de novo somatic mutation cause?

A

Exacerbate ageing and neurological phenotypes

76
Q

What happens in no-dividing cells?

A

Mt DNA is continuously made and destroyed
some replicated more frequently
changes level of heteroplamy

77
Q

how does mtDNA vary in the population?

A

Some varients have remained restricted to certain ethnic groups

78
Q

what is the point mutation in Diabetes and deafness

A

3243 on mtDNA

79
Q

What gene is affected in diabetes and deafness?

A

gene encoding tRNEleu

80
Q

how is diabetes and deafness inerited?

A

maternally

81
Q

how is diabetes and deafness caused?

A

Decreased OXPHOS
decreased ATP
affect insulin production
may affect ion umps required for sound transmission

82
Q

What does Leigh syndrome affect?

A

heart, muscles, eyes. lungs, neurological system

83
Q

How is Leigh syndrome caused?

A

mutations in mtDNA or nuclear genome
affects 4/5 OXPHOS complexes
disordered OXPHOS

84
Q

what mtDNA mutation causes leigh syndrome?

A

Mutation in ATPsynthase subunit

85
Q

What nuclear mutation causes leigh syndrome?

A

Many e.g. COX

Genetics of cell growth and division (4 decks)

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