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Genes and Environment > W11 > Flashcards

W11 Flashcards

1
Q

what does a somatic mutation refer to

A
  • not inheritable
  • only affect somatic cell patches arising from the original cell
  • the earlier in development the mutation occurs, the bigger the patch
  • cancers are often somatic

HOWEVER, risk factors can be inherited

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2
Q

what does a genetic mutation refer to

A
  • occurs in egg and sperm
  • is heritable
  • all cells in the offspring carry the mutation (genotype), but the phenotype is only in cells/tissues usually expressing the gene
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3
Q

what are the inheritance consequences of somatic and genetic mutation

A

somatic mutations are not inherited whilst genetic mutations are

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4
Q

what are examples of somatic and genetic mutation

A

eg. germline mutation: bithorax on Drosophila melanogaster where there is two sets of wings
or antenapaedia: hair of function turns antenna into ectopic legs
or HMA

eg. somatic mutations
patches of colour ie. on hair or a petal of a flower

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5
Q

what are the types of a chromosomal mutation

A

mutations can be chromosomal (large) or affect small number of DNA bases (small)

  • chromosomal mutations can involve many genes and be visible at the light microscope level if it affects >4mb, which contain ~200-300 genes
  • smaller mutations affecting individual genes can occur in coding(affect gene product) or non-coding regions (affect gene expression)

HOW SEVERE THE RESULTING PHENOTYPE IS, DEPENDS ON MANY FACTORS

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6
Q

what are examples of DNA mutation

A

change amount of genetic material:

  1. deletion: may loose important genes
  2. duplication: gene dosage effect

not change amount of genetic material

  1. inversion: disrupt or fuse genes (rearrangement)
  2. translocation: disrupt or fuse gemes (rearrangement)

HOW SEVERE THE RESULTING PHENOTYPE IS, DEPENDS ON MANY FACTORS

can occur anywhere, but the effects described here are assuming these changes occur within the coding sequence

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7
Q

use examples of two different DNA mutations to describe how one might have only little consequence, whilst another may lead to a dramatic phenotype/disease?

A

SINGLE BASE PAIR MUTATIONS:

  • can have different effects on the amino acid polypeptide
    1. silent: (no change in gene product) degeneracy and wobble
    2. missense: (amino acid substitution in gene product) can be first, second or third base
  • could have very little effect on protein function, unless a very important amino acid is lost or the added amino acid changes the 3D protein or binding location
    3. nonsense: (premature termination of translation

DELETION OR INSERTION MUTATION CAN HAVE SUBTLE OR LARGE EFFECTS

  • amino acid deletion/insertion if divisible by three
  • frameshift if not divisible by three
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8
Q

how does that CF mutation affect protein functioning

A
  • there is a defect due tot eh cystic fibrosis transmembrane conductance regulator (CFTR) protein important fir cl- ion transport
  • mutations cause cl- ion to build up which in turns leads to accumulation of sticky mucus in ducts of exocrine, lungs, vas deferens
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9
Q

what are the two key components of haemoglobin

A
  • alpha like genes in chr16
  • beta like genes on chr11
    these have age dependent expression that gives rise to a difference between adult haemoglobin and metal haemoglobin
  • adult 2alpha2beta
  • fetal 2alpha2gamma
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10
Q

how are sickle cell anaemia and thalassemia different

A

sickle cell anaemia cause: 1. one single base pair change in beta haemoglobin leads to a misuse mutation 2. the sixth AA changes from glutamic acid to valine, which alters the charge at this site

thalessemia cause: mutations in alpha or beta thaeleseemia lead to decreased synthesis or stability of either alpha or beta globing respectively. abnormal Hb may lead to excessive RBC loss and cause anaemia.
- autosomal recessive

whilst sickle cell anaemia only has one phenotype of a change in AA, alpha and beta thalessemia have a range of phenotypes respectively
Alpha thalessemia:
1. silent carriers,
2. alpha thalessemia trait (not have ideas yet, one or two alpha genes),
3. Haemoglobin disease- three alpha genes deleted
4. Barts Hydrops fettles syndrome- all foru alpha genes deleted
Beta thalessemia:
1. trait/minor- one gene
2. intermedia reduced - two genes
3. major- 2 genes

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11
Q

what is one type of mutation associated with thalessemia

A

BETA THALASSAEMIA

  1. missense mutants- changes amino acid
  2. nonsense mutants premature stop codon
  3. frameshift mutants
  4. splice mutation: single nucleotide changes may create new splice sites or single nucleotide changes may destroy splice sites and activate cryptic ones
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12
Q

what is a triple repeat mutation

A

changes in phenotype due to expansion of a triplet repeat (3 base sequence)
- the location and type of the expanded repeat varies and results in different outcomes

cases: Huntington’s disease, Fragile X syndrome

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13
Q

use one disease example, how do triple repeat mutations result in a phenotype

A

Fragile X syndrome:

  • Fragile X mental retardation protein is a regulatory protein which binds mRNA in neurons and dendrites
  • synaptic development requires functional FMRP
  • CGG expansion in 5;YTR triggers methylation of promoter and BLOCKS TRANSCRIPTION ie. loss of FMRP

Huntington’s disease:

  • autosomal dominant gain of function
  • CAG repeats in exon 1 of 67 makes protein toxic
  • this leads to clinical symptoms (extra: such as movement disorders, dementia and psychiatric disturbances)
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14
Q

what type of mutation is CF and what is its frequency fact

A
  • autosomal recessive mutation in 7q 31 (chromosome 7 at location 31)
  • there is a high carrier frequency, 1:25. incidence is 1:2500 caucasians
  • the most common mutation is ΔF508: in frame deletion of phenylalanine at position 508
    most common mutation in caucasians:
    DNA change: c.1521_1523delCTT
    amino acid change: Phe508del
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15
Q

how is genetic diagnosis of CF done?

A
  • PCR region of exon 10 with ΔF508 mutation
  • detect deletion on gel: 95 bp versus 98 bp
    Homozygous for ΔF508: only 95 bp
    Heterozygous for ΔF508: both 95bp and 98bp
    homozygous wild type: only 98bp
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16
Q

list CF facts

A
  • CFTR gene cloned in 1989

- currently 2073 mutations identified in this gene

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17
Q

how is CF tested through a Guthrie spot test

A

partial blockage of pancreatic duct leads to increased immunoreactive trpsinogen (IRT)

18
Q

describe beta globin in terms of its gene, mRNA and polypeptide

A
  • gene size of 1.5kb
  • mRNA ~500bp
  • polypeptide contains 146 amino acids
19
Q

what are the effects of the sickle cell mutation

A
  1. Glu to Val causes a defective beta globin which affects haemoglobin structure particularly in hypoxia where the sickle Hb aggregates as rod/rope shaped fibres
  2. this aggregation in turn changes the shape of the RBC to sickle shape, which can block capillaries
  3. the lifespan of RBCs in HbS is reduced from 120-20 days resulting in anaemia
20
Q

which Haemoglobinopathy has varying prevalence across the what and in what way does it vary

A

beta thalassaemia mutations include predictable missense, nonsence, frameshift and less predictable splice mutants: destroy/create site/ activate cryptic site

21
Q

what is Huntington’s disease?

A
  • progressive late onset neurological diseases on chromosome 4
  • homozygosity affects phenotype and disease progression rate
<35 normal
27-35 rare but unstable when transmitted via male
36-39 intermediate (reduced penetrance)
40-50 most adult cases
>50 juvenile onset
22
Q

what is a direct test for Huntington’s disease

A

PCR amplify CAG repeat region of exon 1

<35 normal
27-35 rare but unstable when transmitted via male
36-39 intermediate (reduced penetrance)
40-50 most adult cases
>50 juvenile onset
23
Q

what is Fragile X syndrome

A

when there are fragile sites in human chromosomes

  • there are many people without phenotype
  • though not causative, fragile site on X chromosome correlates with mutation

6-54 normal
55-200 premutation
>200 full mutation

24
Q

how can Fragile X syndrome be detected

A
  • visible when cells cultured in folate deficient thymidine deficient medium
25
Q

what are some facts about inheritance and symptoms of Fragile X syndrome

A
  • most common cause inherited mental retardation and second most common cause of genetic mental deficiency after trisomy 21
  • 1:4000 males, 1:7000 females, 1:130-250 female carrier
  • affected males have behavioural abnormalities, hyperactivity, autistic features, poor eye contact, temper tantrums, IQ<30
  • affected females can have mild learning difficulties to severe retardation
26
Q

what does hybridisation mean and how is it achieved

A

when either RNA or DNA (can be from different sources, synthetic or animal source) are combined with another signal stranded DNA

27
Q

which method do you use to amplify DNA

A

polymerase chain reaction

  • steps are cycled and repeated by chaining temperatures
    1. denaturation of double stranded DNA
    2. annealing/hybridisation of primers
    3. extensiin/elongation by DNA polymerase
28
Q

what key components are required for PCR

A
  • template or orignal target DNA
  • oligonucleotide primers-start and stop of the region
  • deoxyribonucleotide triphosphaes- dNTP- building blocks
  • DNA polymerase enzyme
29
Q

describe the process occurring during each cycle

A

Denaturation:
- dna made single stranded by heating to 95

annealing:

  • primers complementary to ends of the region of interest are annealed at 45-65 degrees
  • primers are 15-30 bases

extension: 70
TAQ polymerase adds new complementary bases to template DNA

30
Q

what is a restriction enzyme

A

enzymes which can cut DNA at specific sequences by recognising specific base sequences: recognition sequences
- it serves to reveal the location of recognition sequences and resulting fragment sizes

31
Q

how do restriction enzymes work and what are they used for

A

there are many different types of restriction enzymes
TYPE 2: cut within and close by this sequence
- cut in three ways: blunt, 5’ overhang or 3’ overhang
note: overhang are called sticky ends, with 5’ or 3’ indicating which of the DNA sides are sticking out

note: TYPE 5 restriction enzyme is the CRISPR, utilises RNA to target specific sequences

32
Q

how does gel electrophoresis work and what is it used for

A

uses the fact that dna is negatively charged due to negative phosphate groups, thus, will migrate from negative to positive electrode

it allows us to sort dna fragments by size

33
Q

what is a vector

A

carries different lengths of DNA eg. plasmid

34
Q

what are some components in a vector

A
  1. origin if replication
  2. antibiotic resistance for selection
  3. multiple cloning site containing unique restriction enzyme sites

properties:

  • carry <10kb DNA inserts
  • circular DNA (number of plasmid per cell equals the copy number ie. number of plasmids expect to see
  • self replicating
  • can integrate into chromosome
35
Q

what are the key steps in amplifying plasmids

A
  1. digest and cut the source dna/vector with the same restriction enzyme to produce compatible ends
  2. combine the source dna and vector through DNA ligase
  3. transform and grow the host and transformed vector through heat shock/ electroporation
  4. undertake screening/selection through antibiotic resistance or lacZ
36
Q

what determines the gel electrophoresis rate

A
  1. gel concentration: slower in denser gel
  2. voltage across field: slower with lower voltage
  3. buffers used
  4. conformation: faster, the more compact DNA is
  5. standard run containing ladder with known DNA fragment sizes
37
Q

outline how DNA sequences that have been cut with restriction enzymes can be rejoined or religated

A

blunt ends can be joined with any blunt end
- sticky ends can only be joined if the base pairs sticking out match ie. are complementary. the easiest way to ensure this is to use the same restriction enzyme

38
Q

what are the components of gene cloning

A
  1. DNA: genomic, synthetic or amplified
  2. vector: carry different length DNA eg. plasmid
  3. restriction enzymes and dna ligase: cut and join to make recombinant dna
  4. host cell: which vectors replicates
39
Q

what kind of variation can occur in restriction enzymes

A

polymorphism (variations in genome may lead to loss or gain go restriction enzyme site

  • this leads to restriction fragment length polymorphism
  • if an RFLP Is linked (close to) a gene mutation, the RFLP will tend to be co-inherited with of the gene alles

results:
middle restriction site present
middle restriction site absent

40
Q

describe competent host cells in DNA cloning

A

eg. e.coli bacteria

- treated to be primed for transformation

41
Q

describe transformation in DNA cloning

A
  • dna uptake
  • heat shock at 42 degrees
  • or. electroporation

note: not all competent hosts are successfully transformed

42
Q

outline differing screening mechanisms to test for dna recombination

A
  1. antibiotic resistance screening: only hosts containing the vector can grow
    - only hosts containing the vector can grow eg. ampicillin resistance
  2. LacZ screening
    basis: only hosts containing the vector can make blue colonies and this follows the biochemical pathway
    X gal substrate to galactosidase + blue compound with the enzyme beta-galactosidase encoded by LacZ MCs
    note: lacZ is a multiple cloning site
    +any inserted DNA in the MCs disrupts the lacZ gene, only those that took in the plasmid(DNA) will be white
    - this is tested through medium containing antibiotic and X gal

Genes and Environment (12 decks)

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