Topic 4 Flashcards
Huntington’s disease
- autosomal DOMINANT
- gain of function
- progressive degerneration of CNS
mhtt mutation
-expansion of trinucleotide repeat CAG in htt gene
-polyQ (glutamine)
-misfolding => aggregation => loss of neurotransmission
normal < 34 polyQ
HD 42 to > 100
trinucleotide repeats =
- instability of the protein
- temporary stem-loop structures (when back to dsDNA will break or slip)
Chaperone proteins and disease
- chaperone proteins help folding conformation
- when chaperone proteins stop working, disease occurs
Molecular diagnosis of HD is done by…
- amplifying repeat region (CAG) by PCR
- 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
Cystic Fibrosis
autosomal recessive loss of function mutation cftr gene encodes ion channels F508
Frame-shift mutation
addition or deletion of nucleotides (1 or 2)
In frame deletion
deletion of 3 or multiples of 3
Missense mutation
substitution of nucleotide that changes codon of amino acid to another
Nonsense mutation
early termination
splicing mutation
alters splicing pattern
Sickle cell anemia
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
Transgene
gene introduced into a cell or an organism creating a transgenic cell or organism
3 vectors for human gene therapy
- adenovirus (non-integrating; transient) gene expression
- retroviruses (integration spot is random) - can result in host inactivation
- Liposomes (lipid coated vesicles and deliver transgene)
Adenovirus
nonintegrating DNA tumor virus
transient gene expresision
viral replication allow cell to cell transmisson
generates host immune response
Retrovirus
integrates into host genome and transmitted to progeny
integration spot is random and result in host gene inactivation
SCID
severe combined immunodeficiency diseases
-affect lymphocytes and immune system making patients susceptible to infections
ADA-SCID
autosomal recessive disease
defective adenosine deaminase (ADA) alleles (on a single gene)
X-linked SCID
mutation of interleukin receptor-encoding gene (IL2RgC) gene on x chromosome
Treatment of ADA-SCID
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
X-linked SCID somatic gene therapy
IL2Rc gene cloned and inserted into retroviral vector
- isolate bone marrow stemcells
- infect bone marrow cell with IL2Rc retroviral vector
- grow cells in culture
- infuse stem cells that express IL2Rc transgene into patient
Disadvantage of IL2Rc transgene
random integration near LMO2 gene which is associated with T cell acute leukemia
because of sequences of IL2Rc near it and activated LMO2
Gene replacement therapy
homologous recombination to replace defective gene with functional
Genome polymorphism
huge number sequences with variations bewteen individuals
Polymorphic sites 4 examples
RFLP
VNTR minisatellites
STR microsatellites
SNP
DNA profiling / fingerprinting and polymorphisms
Polymorphic sites: tandem repeat sites VNTR STR varies between individuals
VNTR and STR (CODIS STR)
-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
SNP
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
Transgenic
organisms with transgenes
Knock out
-site directed recombination
-integrating DNA into a gene (random)
using transgenes
Knock down expression
RNAi against gene of interest
using transgenes
Transgenic animals
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)
Transgenic plants
totipotent - de/redifferentiate
- use of Ti plasmid (tumor inducing plasmid)
- electroporation and gene gun
Tumor induction by Ti plasmid:
- integrates into plant genome
- causes plant cells near site of infection to proliferate forming crown gall
T DNA
transferred DNA
T DNA is located on Ti plasmid and integrated into plant genome at random location
vir region (virulence)
genes required on Ti plasmid for T-DNA excision, transfer and integration into host genome
