Topic 4

Question 1

Huntington’s disease

Answer 1

• autosomal DOMINANT
• gain of function
• progressive degerneration of CNS

Question 2

mhtt mutation

Answer 2

-expansion of trinucleotide repeat CAG in htt gene
-polyQ (glutamine)
-misfolding => aggregation => loss of neurotransmission
normal < 34 polyQ
HD 42 to > 100

Question 3

trinucleotide repeats =

Answer 3

• instability of the protein

- temporary stem-loop structures (when back to dsDNA will break or slip)

Question 4

Chaperone proteins and disease

Answer 4

• chaperone proteins help folding conformation

- when chaperone proteins stop working, disease occurs

Question 5

Molecular diagnosis of HD is done by…

Answer 5

1. amplifying repeat region (CAG) by PCR
2. determine repeat number by separating products on gel
   less than 35 CAG is normal
   >42 is mutant
   rows in gel indicate number of repeats (upper one is longer - 48 CAG)
   Huntington’s is dominant so if even one band appears in the 48 region they will be affected

Question 6

Cystic Fibrosis

Answer 6

autosomal recessive
loss of function mutation
cftr gene
encodes ion channels
F508

Question 7

Frame-shift mutation

Answer 7

addition or deletion of nucleotides (1 or 2)

Question 8

In frame deletion

Answer 8

deletion of 3 or multiples of 3

Question 9

Missense mutation

Answer 9

substitution of nucleotide that changes codon of amino acid to another

Question 10

Nonsense mutation

Answer 10

early termination

Question 11

splicing mutation

Answer 11

alters splicing pattern

Question 12

Sickle cell anemia

Answer 12

T to A mutation in beta globin gene
removal of mstI cleavage
amplify with PCR
dgest with RE
separate gel electrophoresis
ethidium bromide and view with UV light

Question 13

Transgene

Answer 13

gene introduced into a cell or an organism creating a transgenic cell or organism

Question 14

3 vectors for human gene therapy

Answer 14

1. adenovirus (non-integrating; transient) gene expression
2. retroviruses (integration spot is random) - can result in host inactivation
3. Liposomes (lipid coated vesicles and deliver transgene)

Question 15

Adenovirus

Answer 15

nonintegrating DNA tumor virus
transient gene expresision
viral replication allow cell to cell transmisson
generates host immune response

Question 16

Retrovirus

Answer 16

integrates into host genome and transmitted to progeny

integration spot is random and result in host gene inactivation

Question 17

SCID

Answer 17

severe combined immunodeficiency diseases

-affect lymphocytes and immune system making patients susceptible to infections

Question 18

ADA-SCID

Answer 18

autosomal recessive disease

defective adenosine deaminase (ADA) alleles (on a single gene)

Question 19

X-linked SCID

Answer 19

mutation of interleukin receptor-encoding gene (IL2RgC) gene on x chromosome

Question 20

Treatment of ADA-SCID

Answer 20

white blood cell obtained and manipulated and reintroduced
disadvantage: integration site in the genome is random
retroviral vector could activate nearby oncogene and lead to cancer

Question 21

X-linked SCID somatic gene therapy

Answer 21

IL2Rc gene cloned and inserted into retroviral vector

1. isolate bone marrow stemcells
2. infect bone marrow cell with IL2Rc retroviral vector
3. grow cells in culture
4. infuse stem cells that express IL2Rc transgene into patient

Question 22

Disadvantage of IL2Rc transgene

Answer 22

random integration near LMO2 gene which is associated with T cell acute leukemia
because of sequences of IL2Rc near it and activated LMO2

Question 23

Gene replacement therapy

Answer 23

homologous recombination to replace defective gene with functional

Question 24

Genome polymorphism

Answer 24

huge number sequences with variations bewteen individuals

Question 25

Polymorphic sites 4 examples

Answer 25

RFLP
VNTR minisatellites
STR microsatellites
SNP

Question 26

DNA profiling / fingerprinting and polymorphisms

Answer 26

Polymorphic sites: 
tandem repeat sites
VNTR
STR 
varies between individuals

Question 27

VNTR and STR (CODIS STR)

Answer 27

-repetitive DNA elements outside of genes
-unstable
-analyzed by PCR or southern blot
-digest wit restriction endonucleases and separate
-PCR analysis: look at the fluorescence at each locus and compare where the allele is located.
CODIS STRs

Question 28

SNP

Answer 28

single-nucleotide polymorphisms
-single base pair substitution in reference to a sequnce
-noncoding region
haplotype - set of SNP on same chromosome – inherited together
-track evolutionary history

Question 29

Transgenic

Answer 29

organisms with transgenes

Question 30

Knock out

Answer 30

-site directed recombination
-integrating DNA into a gene (random)
using transgenes

Question 31

Knock down expression

Answer 31

RNAi against gene of interest

using transgenes

Question 32

Transgenic animals

Answer 32

ES obtained from black
ES embryonic stem cells are pluripotent (two different ES)
Injected into blastocyst of mouse that becomes pseudopregnant (white)
Offpsring: chimera (spotted)
Dark coloured offspring (second generation)

Question 33

Transgenic plants

Answer 33

totipotent - de/redifferentiate

• use of Ti plasmid (tumor inducing plasmid)
• electroporation and gene gun

Question 34

Tumor induction by Ti plasmid:

Answer 34

• integrates into plant genome

- causes plant cells near site of infection to proliferate forming crown gall

Question 35

T DNA

Answer 35

transferred DNA

T DNA is located on Ti plasmid and integrated into plant genome at random location

Question 36

vir region (virulence)

Answer 36

genes required on Ti plasmid for T-DNA excision, transfer and integration into host genome