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genomics notes > mitochondrial genome > Flashcards

mitochondrial genome Flashcards

1
Q

What are features of mitochondrial DNA (mtDNA)?

A
  • circular, double stranded and smaller than one chromosome.
  • multi-copy
  • 37 genes, oxidative phosphorylation protein subunits, transfer RNAs and ribosomal RNAs.
  • no introns
  • D loop is non -coding. Replication (origin of heavy strand) and transcription (heavy/light strand promoter) is initiated here.
  • maternally inherited. No recombination.
  • packaged into nucleotides
  • mtDNA haplogroups can show us human migration patterns.
  • mtDNA is a hexamer - unwinds double stranded mtDNA to allow for replication by polymerase gamma.
  • mitochondria single stranded binding protein (mtSSBP) binds to single strand DNA and protects nucleases and prevents secondary structure formation. stimulates twinkle (mitochondrial protein) to enhance synthesis.
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2
Q

Does heavy strands have more of purine or pyrimidines?

A
  • heavy has more purines (Adenine and guanine)

- light has ore pyrimidines (thymine and cytosine)

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3
Q

Which enzyme complex does mitochondrial genome code for?

A
  • 1, 3, 4, 5

- only complex 2 is soley nuclear

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4
Q

Which transcription factor acts as a histone?

A
  • transcription factor A
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5
Q

What are the exceptions to universal genetic code in vertebrate mitochondria?

A
  • methionine(AUG , AUA)
  • tryptophan (UGA)
  • stop codons(AGA, AGG)
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6
Q

Describe replication in the mitochondria?

A
  • requires nuclear and mtDNA coded proteins.

- all proteins involved in replication/transcription/ translation are imported into mitochondria.

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7
Q

How many catalytic and accessory subunits does polymerase gamma have?

A
  • one catalytic subunit

- two accessory subunits

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8
Q

What is the function of polymerase gamma A?

A
  • 3’-5’ exons lease domain proofreads
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9
Q

What is the function of polymerase gamma B?

A
  • interacts with DNA template and increases activity polymerase gamma A.
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10
Q

outline the strand displacement model.

A
  1. parental heavy strand displayed and coated with mtSSBP.
  2. Twinkle helical unwinds mtDNA
  3. mitochondrial RNA polymerase (POLMT) synthesis RNA primer using light strand as template.
  4. POLy (gamma polymerase) uses primer to replicate DNA at origin of heavy strand
  5. Heavy strand replication passes origin of light strand
  6. stem loop structure formed preventing mtSSBP binding.
  7. POLRMT synthesises primer using heavy strand as template and POLy ses primer as template to replicate light strand at origin of light strand
  8. continues till both strands fully replicated
  9. daughter molecules segregated.
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11
Q

How is mitochondrial DNA different from nucleus DNA?

A
  • mitochondrial DNA is inherited from mother unlike nucleus DNA which is inherited from both parents.
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12
Q

What does the non- coding region contain?

A
  • regulatory sequences for replication and transcription
  • mtDNA replication starts in origin of heavy strand.
  • transcription starts at heavy strand promoter (HSP) and light strand promoter( LSP)
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13
Q

How is mtDNA packaged?

A
  • mtDNA is packaged into structures called nucleoids
  • one or two copies of mtDNA per nucleoid
  • transcription factor A (TFAM) acts as a histone protein.
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14
Q

What are exceptions to the universal genetic code?

A

=> genetic code in vertebrate mitochondria:

  • AUA and AUG for methionine (AUA codes for isoleucine in nuclear DNA)
  • UGA codes for tryphtophan (UGA is a stop codon in nuclear DNA)
  • AGA and AGG are stop codons in mitochondria (not arginine)
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15
Q

What are the 3 mitochondrial

DNA polymerase?

A
  • polymerase gamma
    => heterotrimer protein
    => one catalytic subunit (polymerase gamma A)
    => two accessory subunits (polymerase B)
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16
Q

What is the function of polymerase A?

A
  • contains 3’-5’ exonuclease domain to proofread newly synthesised DNA
17
Q

What is the function of polymerase B ?

A
  • enhances interactions with DNA template and increases activity and processivity of polymerase gamma A
18
Q

What is mitochondrial DNA helicase called and what is its function?

A

=>TWINKLE

  • hexamer - 6 twinkle subunits
  • unwinds double stranded mtDNA template to allow replication by polymerase gama
19
Q

What is the function of mitochondrial single stranded binding protein (mtSSBP)?

A

=> binds to single stranded DNA

  • protects against nucleases
  • prevents secondary structure formation
  • enhances mtDNA synthesis by stimulating TWINKLE helicase activity
20
Q

outline the steps in the strand displacement model of mtDNA?

A
  1. replication of heavy
  2. replication of light strand begins O
  3. replication
  4. segregation of daughter cells
21
Q

What is the role of Top3a (top 3 alpha)?

A
  • lack of Top3a in cells with those mitochondria showed concatenation (mitochondrial DNA was not able to segregate)
22
Q

What are examples of some mitochondrial syndromes?

A
  • Leigh syndrome = most common >80 genes linked to the disease.
  • LHON = leber’s heridity optic neuroretinopathy
    -KSS = Kearns - Sayre syndrome
    MELAS = mitochondrial encephalomyopathy lactic acidosis stroke like episode.
    MERFF = myoclonus eplilespsy red ragge fibers
    NARP = neurogenic muscle weakness Ataxia Retinintis
    MINGIE = mitochondrial myopathy NeuropathyGastrointestinal disease
    => majority of ppl wont display these syndromes so careful clinical assessment is required.
23
Q

Diagnosis of mitochondrial disease?

A
  • clinical signs
  • blood and tissue histochemical and analyte measurements
  • neuroimaging
  • enzymatic assays of OXPHOS in tissue samples and cultured cells
  • DNA analysis.
24
Q

What are some histochemical staining of muscle mitochondria and COX/SDH?

A
  1. Gomori trichome red
    - used to identify ragged red fibres, which are muscle cells characterised by accumulation of mitochondria which is the cellular response of the oxidative phosphorylation response.
  2. SDH (complex II)
  3. COX (complex IV)
25
Q

What method is used to measure OXPHOS activity?

A
  • spectrophotometry of OXPHOS activity

- high resolution respirometry measures oxygen consumption.

26
Q

How can defects in OXPHOS complexes be measured?

A
  • blue - native gel electrophoresis
  • larger complexes found higher up in the gel.
  • deficiency of one protein can cause loss of the whole complex
27
Q

Why is there a change in architecture of mitochondria when there are defects?

A
  • as a result of remodelling of the lipid membrane of mitochondria
  • cant be seen using light microscopy, only seen via electron microscopy.
  • appear large and swollen, and paracrystalline inclusions (PCI) seen.
28
Q

What was the first reported mitochondrial disease?

A
  • 30 y/o woman
  • age of onset 8 years (disease started at 8)
  • high energy intake 3000 kCal per day
  • stable 38kg 159c,
    => myopathy
29
Q

How is mitochondrial DNA inherited?

A
  • maternally inherited.
  • males affected by disease do not pass on MD.
  • point mutation at M8969 from G -> A
30
Q

What are 2 types of cellular population of mitochondria DNA

A

=> homoplasmy = presence of only 1 single mtDNA variatnt

=> heteroplasmy = presence of 2 or more mtDNA variant.

31
Q

How does heteroplasmy levels determine disease?

A
  • relative % of pathogenic mtDNA determine if disease manifests or not.
  • 80% or more variant leads to disease manifestation.
32
Q

How does heteroplasmy levels affect penetrance and severity of disease?

A

=> m8993 T>G

  • > 90% = leigh syndrome
  • 60-75 % = NARP
  • < 60% asymptomatic

=> variable penetrance also in homoplasmic mutations

  • LHON
  • 50% males affected
  • 10% females affected

genomics notes (18 decks)

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