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Sem 1: Genetics > Week 12 > Flashcards

Week 12 Flashcards

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Biology 101 flashcards
1
Q

What is the electron transport chain?

A

Theelectron transport chainis a collection of membrane-embedded proteins and organic molecules, most of them organized into four large complexes labeled I to IV.

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2
Q

What happens in the electron transport chain to produce energy?

A

As the electrons travel through the chain, they go from a higher to a lower energy level, moving from less electron-hungry to more electron-hungry molecules.

Energy is released in these “downhill” electron transfers, and several of the protein complexes use the released energy to pump protons from the mitochondrial matrix to the intermembrane space, forming a proton gradient.

In the inner mitochondrial membrane, H^++start superscript, plus, end superscriptions have just one channel available: a membrane-spanning protein known asATP synthase.

Conceptually, ATP synthase is a lot like a turbine in a hydroelectric power plant. Instead of being turned by water, it’s turned by the flow of H^++start superscript, plus, end superscriptions moving down their electrochemical gradient.

As ATP synthase turns, it catalyzes the addition of a phosphate to ADP, capturing energy from the proton gradient as ATP.

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3
Q

What is unique for mechanism and patterns of inheritence?

A

Inheritance of cytoplasmic genomes is distinct from nuclear genomes (in animals they are mitochondrially inherited)

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4
Q

Where are cytoplasmic genomes are located?

A

Mitochondria (animals, plants and fungi)
Chloroplasts (plants)

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5
Q

What is required for biogenesis of functional mitochondria?

A

It is dependant on co-ordinated expression of nuclear and cytoplasmic genomes

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6
Q

What are the different genes in the mitochondria?

A

2 rRNAs
22 tRNAs
13 protein genes

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7
Q

What happens if there a mutation in mtDNA?

A

They are mostly lethal

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8
Q

What is the relationship between mt genome and mt rRNAs?

A

Mitochondria have their own ribosomes but while mitochondrial rRNAs are encoded in themitochondrial genome, the proteins that make up mitoribosomes are encoded in thenucleusand assembled by cytoplasmic ribosomes before being implanted into the mitochondria
55S (39S/28S) and 80 proteins as opposed to the 80S eukaryote mitochondria

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9
Q

How did they demostrate the maternal inheritance of animal mtDNA?

A

The mtDNA of X(Xenopus). laevis and X. borealis can be distinguished at the molecular level.
Reciprocal crosses were performed and the mtDNA genotype of the F1 progeny was determined.
These analyses demonstrated maternal inheritance of mtDNA in Xenopus.

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10
Q

Why is mitochondrial DNA inherited maternally?

A

Differences in gamete size means zygotes would receive very few paternal mitochondria.
Paternal mitochondria do not normally enter the egg (chemically marked and degraded if they do).
Human eggs contain c.2000 mitochondria.

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11
Q

What is the structure of mitochondrial genome?

A

Thehuman mitochondrial genomeis a circular double-strandedDNAmolecule of about 16kilobases
As in prokaryotes, there is a very high proportion of coding DNA and an absence of repeats

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12
Q

How are mitochondrial genes transcribed?

A

Mitochondrial genes are transcribed as multigenic transcripts, which are cleaved and polyadenylated to yield mature mRNAs

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13
Q

Where are most genes for mitochondria encoded?

A

Most proteins necessary for mitochondrial function are encoded by genes in the cell nucleus and the corresponding proteins are imported into the mitochondrion
NADH dehydrogenase
Cytochrome
Part of the electron transport chain for making ATP

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14
Q

What is different about the genomone DNA and mitocondrial DNA sequence?

A

Researchers observed differences in mitochondrial proteins predicted from the gene sequence
UGA is stop codon in universal code but codes for Trp in mt code
AGG/AGA is Arg in unviversal code but codes for stop codon in mtDNA code

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15
Q

What can variations in the genetic code show?

A

Determine relative evolutionary proximity to other species

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16
Q

When did the first mitochondria appear?

A

Around 1.6 bya originating as an aerobic prokaryote in a symbiotic relationship with anaerobic eukaryote

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17
Q

What is another cause for mitochondrial disorders outside of mitochondrial mutations?

A

Mitochondrial disorders can be a consequence of nuclear gene mutations that encode mitochondrial proteins. These mutations show strictly Mendelian patterns of inheritance (usually autosomal recessive). In total over 200 genes have been identified

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18
Q

What is the overview of mitochondrial diseases?

A

Several diseases of the human nervous system are caused by mutations in the mitochondrial genome which shows a distinct pattern of inheritance.
Mutations are passed from mothers to children (maternal transmission).
Symptoms vary due to heteroplasmy

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19
Q

What is heteroplasmy?

A

Variation in the types of mitochondria (some mutant some wildtype)

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20
Q

How does heteroplasmy cause variation of symptoms?

A

Different split of mitochondria therefore some oocytes have more mutant mitochondira which causes high levels of symptoms also the opposite could occur with low level symptoms

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21
Q

What is the Charlie Gard case?

A

The legal battle that centered around Charlie Gard captured international attention and offers of support from Donald Trump and the Pope
Disease is called infantile onset encephalomyopathy mitochondrial DNA depletion syndrome (MDDS).

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22
Q

What are the symptoms of MDDS?

A

MDDS causes progressive muscle weakness and brain damage

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23
Q

What causes MDDS?

A

Autosomal recessive nuclear mutation in the RRM2B gene (Chromosome 8)
RRM2B encodes a ribonucleotide reductase enzyme
It is required for normal mtDNA synthesis and repair
These parents must both be heterozygous carriers of the nuclear mutation

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24
Q

Where can mitochondrial DNA mutations impact?

A

They can affect everywhere:
Heart- Cardiomyopathy
Pancreas- Diabetes
Brain- Seizures
Skeletal muscle- Myopathy

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25
Q

How many mitochondria do human cells contain?

A

100s of mitochondria each with 2-10 copies of mtDNA

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26
Q

Why does mtDNA mutate quickier than nDNA?

A

Oxidative phosphorylation system in the mitochondria generates free radicals, which can damage DNA.
Any impairment of mt function can lead to the accumulation of reactive oxygen radicals which can damage DNA.
MtDNA replication and repair is not as efficient as nuclear DNA
Mt is like a biological nuclear powerplant

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27
Q

What tissues are mostly affected by mitochondrial diseases?

A

High energy tissues like nervous and muscle tissues

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28
Q

What is a key feature of mitochondrial mutations?

A

Defective mitochondria leding to muscle degeneration
Common symptoms of mitochondrial dysfunction (diabetes, dementia, muscle weakness, loss of sight and hearing) are also characteristic of the aging process suggesting somatic mtDNA mutations accumulate over time.

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29
Q

What is a consequence of mtDNA?

A

Accumulation of mtDNA mutations overtime nat result in an age-related decline in oxidative phosphorylation

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30
Q

What evidence for mtDNA and aging?

A

Percentage of heart tissues with a mitochondrial deletion increases with age.
Brain cells of people with Alzheimer’s Disease (AD) have abnormally low energy metabolism.
20% to 35% of mitochondria in brain cells of most AD patients have mutations in cytochrome c oxidase genes, which may explain their low energy metabolis

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31
Q

What are examples of deletions in mtDNA causing mt disease?

A

CPEO- Chronic progressive external opthalmoplegia - leads to paralysis of eye muscles
KSS- Kearns-Sayre syndrome - retinal deterioration heart disease, hearing loss, kidney failure

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32
Q

What causes pearson’s syndrome?

A

Bone marrow dysfunction, pancreatic failure
G to A mutation resulting in Arg to His substitution in the ND4 gene for complex 1

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33
Q

What causes myoclinic epliepsy and ragged red fibre disease (MERFF)?

A

Mutation in the tRNA gene affects components of NADH deHydrogenase complex 1 and cytochrome oxidase complex IV)

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34
Q

What causes up to 60% of mitochondrial disease?

A

Up to 60% of mitochondrial CPEO are due to mitochondrial DNA (mtDNA) deletions (ranging form 1.3 to 1.9 kb)

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35
Q

What is another major cause of mitochondrial disease?

A

Are caused by nuclear DNA related defects of mtDNA maintenance eg POLG1 and ANT

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36
Q

What mutations cause MERFF?

A

m.A8344G, m.T8356C, m.G8361A, and m.G8363A. The point mutation m.A8344G is most commonly associated with MERRF, nucleotide 8344 (A8344G) of the tRNA(Lys) gene of mitochondrial DNA (mtDNA)

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37
Q

What are the symptoms of MERFF?

A

Symptoms include ataxia (muscle weakness), deafness, dementia and epilepsy.
Defective mitochondria leads to decreased ATP generation –> muscle degeneration
The severity is dependant on heteroplasmy

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38
Q

What are maternal sufferers of MERFF?

A

They are heteroplasmic for wtMtDNA and mutant with a single base substitution in tRNA.

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39
Q

What is the threshold level for A8344G mutation required in mother for all children to inherite the disease?

A

35-40%

40
Q

What is the relationship between high energy tissues and tolerance for mutant mitochondria?

A

Tissues with higher energy requirement are less tolerant of mutant mitochondria

41
Q

When are low energy tissues impacted by mutant mitochondria?

A

When the proportion of wild-type mitochondria is greatly reduced

42
Q

What % of mutant mitochondria in different tissues is the threshold for MERFF symptoms?

A

Brain- 20%
Heart- 40%
Skeletal muscle type 1- 60%
Skeletal muscle type 2- 80%
Skin - 80%

43
Q

What are the 2 forms of mitochondrial genome replacement therapies?

A

Spindle transfer and pronuclear transfer

44
Q

What is the method for spindle transfer?

A

mtDNA donor oocyte has karyoplast discarded
Patient oocyte with mutated mtDNA has cytoplast removed
Karyoplast (nDNA transfered) is added to the donor cytoplast
Recontructed oocyte is fertilised
Embryo implanted into patient

45
Q

What is the method for pronuclear transfer?

A

mtDNA donor oocyte fertilised and then the karyotype is discarded
Patient oocyte is fertilised and the the cytoplast is discarded
Karyoplast (nDNA transfered) is added to the donor cytoplast
Embryo implanted into patient

46
Q

What is controversial about pronuclear transfer?

A

Requires discarding a fertilised egg

47
Q

Whats the overview of the first spindle transfer performed on primated?

A

Rhesus monkies in 2009 - Mito and Tracker were fraternal twins
Each contain nuclear DNA from their mother and father and mtDNA from the oocycte donor
Confirmed by molecular analysis

48
Q

At what stage is the maternal nDNA transfered into the donor cytoplast?

A

Maternal nDNA is transferred in the form of the mitotic spindle (ST) from oocytes at metaphase II of meiosis

49
Q

What is the case study of using spindle transfer technology in humans?

A

The Jordanian parents first two children died form leigh syndrome (20% cases are due to mtDNA mutations)
Completed using spindle transfer as parents objected to destroying a fertilised egg produced via PNT route
OPeration carried out in mexico by US doctors

50
Q

What is a new technology to help cure mtDNA diseases?

A

They can use viruses (adeno-assocated virus (AAV) serotype) to add a promoter and the required genes in order for the cell to produce the proteins required for normal mitochondria function

51
Q

What was the traditional approach for gene knockout?

A

Trhough the use of Homologous recombination

52
Q

What is requried for homologous recombination?

A

2 500 base pair homologous arms

53
Q

What is the difference between homologous section on plasmid compared to the bacterial genome?

A

The plasmid lacks the targeted gene in the middle meaning if integration occurs it will cause a gene knockout

54
Q

What is the intergration step of homologous recombination?

A

The entire plasmid has been integrated through either but not both of the homologus regions

55
Q

What is the resolution step of homologous recombination?

A

There are two forms of resolution:
1- It resolves it self so that only the plasmid intergration has occured. so you get the intergration causing gene knockout
2- Target gene has been left it meaning plasmid insertion has failed

56
Q

What are the disadvantages of using homologous recombination?

A

Low “hit” rate and labour intensive but large numbers of bacteria can be grown

57
Q

What can be used to help find sucessful recombinants?

A

Selection markers postive or negative

58
Q

What is a positive selection marker?

A

A marker that helps bacteria eg antibiotic reistance gene like Amp resistance

59
Q

What is a negative selection marker?

A

Helps determine whether a bacteria has properly intergrated the section of plasmid DNA rather than the entire plasmid. This is done by creating a gene that actively harms the bacteria as seen in temperature sensitive ori

60
Q

When was homologous arm first developed?

A

1989

61
Q

How does homologous recombination work in mice?

A

Similar method than with bacteria. Thisis done through using a targeting vector and the homologous recombination of specific exons. Electroporation allowing for the targeting vector to get across the transport membrane

62
Q

What is the positive selection marker in homologous recombination of mice?

A

Neo^r-= resistance gene
Neomycin = antibiotic that is toxic to mammalian cell cultures

63
Q

What is the negative selection marker in homologous recombination of mice?

A

HSV-tk = encodes an enzyme that converts antigerpetic drugs into toxic DNA replication inhibitors

64
Q

When was homologous recombination in mice recorded?

A

In 1989 and won the Nobel prize in Physiology or Medicine

65
Q

What was homolohous recombination in mice used for?

A

Genetics of cancer, obesity, heart disease, substance abuse, Parkinson’s, ageing, stress

66
Q

What was the work of Professor Maria Jasin?

A

She theorised that HR might play a role in DSB repair- Homology dependent repair (HDR)
She proved this in the 80s first in yeast then in mice, usng a “rare-cutter” restriction enzyme Isce-1

67
Q

What is useful about DSB?

A

Sites of dsDNA break repair were editogenic and could be used for site-specific gene knock-out or knock-in

68
Q

How much more common is homologous repair at DSBs then without?

A

HR is 100x more common at DSB sites with lower non canonical integration is much lower

69
Q

What is the process for Homology-directed repair?

A

A broken double strand has occured and in certain parts of cell cycle it can look for homologous DNA sequences
Homologous DNA sequence is found a strand invasion occurs.
The broken strand invades the template strand and forms a D loop, which keeps the two strands apart.
The broken strand repairs itself with whatever is in the sister chromatid
It then rejoins up with either a function version of gene or an introduction of required gene

70
Q

What is the recognition site for Isce-1?

A

5’ to 3’
TAGGGATAACAGGGTAAT

71
Q

When does HDR occur?

A

S or G2 phase and every cell has a different length (gametes have extended phases) the rest of time only NHEJ occurs

72
Q

What are zinc finger domains?

A

Are small protein motifs that contain multiple finger-like protrusions that make contact with DNA molecules

73
Q

Where were zinc fingers first identified?

A

As part of transcription factors in african clawed frog (Xenopus laevis)

74
Q

What is the natural function of zinc fingers?

A

The binding strength of transcription factor was due to the prescence of the zinc finger structures

75
Q

How are zinc fingers made more versitile?

A

Protien engineering techniques can be used to alter the DNA-binding specificity. Tandem repeats can produce targeted DNA binding

76
Q

What is Fok-1?

A

It is a type IIS (II=2) restriction enzyme

77
Q

What is a type II restriction enzyme?

A

It recognises a base sequence then cuts an ‘n’ number of bases away

78
Q

What is Zinc FInger Nucleases (ZFN)?

A

Protein engineered Zinc finger motif designed to recognise a specific 3 base DNA sequence fused with he the cleavage domain of Fok1

79
Q

What is requried for a ZFN to create a double break?

A

Each ZFN produces a single strand break - pair required for dsDNA break

80
Q

When have ZFN been used?

A

They demonstrated in 2006 to generate spontaneous yellow mutants in Drosophila melanogaster

81
Q

What was the idea for using gene editing as a cure for HIV?

A

Early 1990s - few promising treatments of HIV
Naturally occuring vairent of the Varient- delta32 appeared to comfer resistance
Cohort studies found no homozygote HIV patients

82
Q

How frequent is delta32 in european genotypes?

A

10% with 1-2% homozygote

83
Q

What are the advatages of ZFN?

A

High specificity
Low immunity
Currently being used in clinical trial

84
Q

What are the limitations of ZFN?

A

Non-modular - difficult and labourious engineering process
Difficult to predict cytotoxic offshoots
Some nucleotide triplets do not have a corresponding zinc finger

85
Q

What is the overview of Transcription Activator-Like Effector Nucleases (TALENs)?

A

Fok1 endonuclease fused to TALE domains, which interact with target DNA
1 TALE domain recognises 1 DNA base pair
Single strand cleavage

86
Q

What is the advantages of TALENS?

A

Modular assembly
Fusing multiple TALE domains together does not effect binding specificity
Similar targeting effciency with less cytotoxic effects

87
Q

What are the limitations of TALENS?

A

5’ base of TALEN target site must be a thymine
OFf target effects
TALEN binding is negatively impacted by DNA mehtylation

88
Q

What is the overview of CRISPR?

A

Cas9 endonuclease, derived from Streptococcus pyogenes, rescruited to DNA with a sgRNA
Site specificity results from 20 base pairs of sgRNA interacting with target DNA via Watson-Crick base pairing
Double strand break

89
Q

What are the advantages of CRISPR?

A

Easy to generate via simple molecular cloning techniques
Higher target effciency
Ability to multiplex for targeting multiple genes

90
Q

What are the limitations of CRISPR?

A

Requirement for a PAM sequence to target site
Higher off target effects
Binding effciency impacted by chromatin accessibility

91
Q

What is the function of Cas13?

A

It targets mature mRNA degrading it resulting in gene knockdowns not knockouts

92
Q

What is dCas9-Fok1?

A

It is the binding of Fok1 nucleases with Cas9. The Cas9 has the cutting region removed

93
Q

What is the function of dCas9?

A

It sits of DNA at a specific location which prevents DNA transcription

94
Q

What is the advantage of dCas9-Fok1?

A

Requires two molecules to break DNA so reduces off cutting and can increase efficiency

95
Q

What is tne advantage of Cas12?

A

DSB are staggered to reduce the chance of NHEJ

96
Q

What is Anti-Cas?

A

Proteins that supress Cas9 molecules reducing off cutting by supressing Cas9 synthesis

Sem 1: Genetics (10 decks)

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