the mitochondrial genome

Question 1

describe the mitochondrial genome

Answer 1

double stranded circular molecule

consists of heavy and light strand

multi copy genome (10-100,000 copies per cell)

37 genes

no introns

d-loop= non coding region where replication and transcription are initiated

maternally inherited, no recombination

Question 2

what’s the non coding region contain

Answer 2

contains regulatory sequences for replication and transcription

mtDNA replication starts in origin of heavy strand

transcription starts at heavy stand promoter and light strand promoter

Question 3

how is mtDNA packaged

Answer 3

into structures called nucleoids
one or two copies of mtDNA per nucleoid
transcription factor A acts as histone protein

Question 4

exceptions to the universal genetic code

Answer 4

genetic code in vertebrate mitochondria

AUA and aug for methionine
uga codes for tryptophan
AGA and AGG are stop codons

Question 5

mitochondria requires both nuclear and mtDNA encoded proteins

Answer 5

encodes 13 proteins of OXPHOS

to make 13 OXPHOS proteins mtdna must be:
replicated
transcribed
translated

all proteins involved in replication translation and transcription of mtDNA are encoded by nuclear genes and imported into mitochondria

Question 6

mitochondrial DNA polymerase

Answer 6

polymerase gamma
heterotrimer protein
polyA contain 3-5’ exonuclease domain to proofread newly synthesised DNA

poly B enhances interactions with dna template and increase activity and processivity of polyA

Question 7

mitochondrial DNA helices twinkle

Answer 7

twinkle = hexamer, six twinkle subunits

unwinds double stranded mtDNA template to allow replication by poly

Question 8

mitochondrial single stranded binding protein

Answer 8

binds to ssDNA
protects against nucleases
prevents secondary structure formation
enhances mtDNA synthesis by stimulating twinkle helicase activity

Question 9

strand displacement model of mtDNA replication

Answer 9

parental heavy strand displaced and coated with mtssbp
helicase unwinds mtDNA
mitochondrial RNA polymerase synthesises RNA primer using light strand as template poly uses RNA primer to replicate DNA at oh

Question 10

strand displacement model of mtDNA replication 2

Answer 10

heavy strand replication passes OL
stem loop structure is formed preventing mtssbp binding
mitochondrial RNA polymerase synthesises RNA primer using heavy strand as poly uses RNA primer to replicate light strand at Ol

Question 11

mitochondrial syndromes

Answer 11

Leigh syndrome 
lebers hereditary optic neuroretinopathy
Kearns Sayre syndrome 
MELAS
MERFF
NARP
MINGIE

Question 12

diagnosis of mitochondrial disease

Answer 12

clinical signs
blood and tissue histochemical and analyse measurements
neuroimaging
enzymatic assays of OXPHOS in tissue samples and cultured cells
DNA analysis

Question 13

low invasive biochemical investigations

Answer 13

blood/CSF lactic acid >2.1 mM
lactic acid/pyruvate ratio
amino acids
organic acids

Question 14

muscle histology

Answer 14

haematoxylin and eosin
gomori trichrome 
SDH
COX
combined COX/SDH

Question 15

MtDNA genome sequencing

Answer 15

blood, urine, fibroblasts, tissue
next gen sequencing of mtDNA
quantitative PCR for mtDNA depletion

Question 16

mutations in mtDNA replication machinery cause secondary mutations in mtDNA

Answer 16

mtDNA deletions
mtDNA depletion
occurs in post-mitotic tissues
brain
muscle
heart 
liver

Question 17

NGS sequencing approaches for nuclear genes

Answer 17

sequencing panels of disease specific genes

whole exome sequencing

whole genome sequencing

Question 18

genetic counselling for mtDNA mutations

Answer 18

prognosis depends on mutation, heteroplasty levels, variable penetrance of homoplasmic mutations and difficult to predict

recurrence risks
strictly maternally inherited

Question 19

preventing transmission of mtDNA mutations

Answer 19

oocyte donation
prenatal diagnosis
preimplantation genetic diagnosis
mitochondrial replacement therapy