Genetic information

 DNA = deoxyribonucleic acid

 RNA = ribonucleic acid

Structure of 4 bases that comprise DNA

 Purine:
Purine
Adenine Guanine

 Pyrimidines
Pyrimidin
Thymine e Cytosine

H-bonding between bases directs base pairing:

 A always pairs with T
 G always pairs with C

Structure of ds DNA ‘

(DNA) Replication
Gene expression: 1. Base pairing
Nucleotide
 DNA contains instruction on how a cell functions
o Proteins perform these functions
 How are the instructions translated from CODE
nucleotide language of DNA into AA language of
proteins?
o Via an intermediary RNA
o DNARNAprotein
TRANSCRIPTION:
 DNA is transcribed into messenger RNA (mRNA), using
base pairing
o This mRNA = translated into protein
o The code is a triplet code: 3-nucleotide code for
each AA

The genetic code: the codon is on the bottom

 What do the following code for? (CODON: PROTEIN)
o AGC: SER
o GGG: GLY:
o UAG: STOP
o AUG: START

TRNASLATION:
 How is the mRNA translated into protein?
o = there is a ‘go-between” = transfer RNA (tRNA) (b,
c)
 There is a separate tRNA for every AA
o The AA is attached to 1 end of tRNA 2.
o The other end has a triplet code (ANTICODON) translation
that will base-pair with t-RNA 3D structure
mRNA AA at 3’ end

 Translation RNAprotein
o The protein synthesis
factory = called the
ribosome
o tRNA carrying their amino
acid = move to the
ribosome
o they base-pair with mRNA
o this places their AA in the
correct sequence to form
the protein that was coded
in the mRNA (and hence in
DNA sequence)
Anticodon = Biochemistry
in the middle diagram/symbol
of tRNA for t-RNA
 tRNA + binding
f with mRNA
using codon +
Control of gene expression – PROKARYOTES anticodon
 in prokaryotes, control of gene expression
o = occurs at the level of transcription i.e.
whether a gene is transcribed or not
o once the mRNA is formed, it is translated
immediately
 control of gene at level of transcription:
o example:

 e.g. transcriptional regulation in E coli:

o the usual energy + carbon source for E coli is glucose
 when glucose is scarce, lactose can be used
 lactose is a disaccharide, which must be
hydrolyzed before it can be utilized
o the enzyme that catalyzes this reaction is beta-
galactosidase

o beta-galactosidase is only produced when lactose is present

o regulatory elements control the level of expression of genes that code for proteins
 B) lactose operon – e.g. z, y, a = genes coding for enzymes needed to
metabolize lactose + includes promoter + operator site; also repressor site (i)
 A) when repressor bind to operator site = prevents transcription of z, y, a
 D) When induce added to molecule =repressor inducer does not bind to DNA


Control of gene expression – EUKARYOTES
 Eukaryotic cells:
o mRNA is formed as pre-mRNA in the nucleus (where DNA is)
o the pre-mRNA is extensively modified
o it is then exported to the cytoplasm, where protein synthesis
occurs
 e.g. control of gene expression at transcription level in drosophila
o group of cells that gives rise to adult eye, and adult leg
o agene artificially expressed in leg precursor cells
 Transcriptional regulation leads to tissue specific expression of
genes
o BLACK DOTS = Indicate gene expressed in different tissues

Control of Eukaryotic gene expression

 Transcription:
o Transcription factors
o Activators/enhancers
o Epigenetics modifications
 Chromatin remodeling
 Methylation of ‘silent’ genes
 Initiation of transcription in eukaryotes
o Proteins called transcription factors = meditate the
initiation of transcription
o This is an additional control mechanism for gene
expression in eukaryotes
 TATA box RNA polymerase II RNA transcript
(transcription initiation complex)
 Activators are also required for eukaryotic gene transcription
o Transcription in eukaryotes
 The interaction of only a few different activators/inhibitors
can regulate a large no. of genes differentially
  Epigenetic modifications:
o DNA is packaged with proteins in the cell –chromatin. For genes to be transcribed,
the chromatin must be unpacked – this is another level of control

 Methylation of DNA and gene silencing

o Methylation: added a CH3 mythl group –
promotes other proteins to bind to
methylated DNA = to the gene to silence it –
promotes histone deacetylase and chromatin
remodelling complex added to completely
turn the gene off

Control of eukaryotic gene expression:

 Post-transcriptional modification
o Splicing =
o Transport from nucleus to ribosome
o ½ life of mRNA
  Exons can be spliced in different ways to produce different proteins from the same gene
sequence

Introns Exons

oSplicing =cutting out the introns leaving only exons for gene expression, before
gene is transcripted to preRNA
o E.g. Muscle alpha-tropomyosin has 12 exons =
which are spliced to produce different mRNAs in
different tissues
 Summary of transcriptional regulation in eukaryotes
o 5’ end =upstream, 3’ end = downstream.
Enhancers + proximal control elements and
promoters cut of splicing occurs to become
pre-mRNA then become mRNA to start
CODING

Application: microarrays - used to identify tumour/metastasis

 Although the genomes of many organisms have been
sequenced, we still don’t know the function of Many
putative genes or how/when they are expressed
 Microarrays: a technique to study the expression of
thousands of genes in different tissues/cells
=experiment
o Expression of genes = can be in glucose
(green) or in ethanol (red) or both
o This is when cells are grown in the medium, and
then total mRNA is isolated and reverse
transcribed to cDNA labelled with a Fl dye \
 Genes may be over- or under- expressed
o Red= over expressed, green under expressed
o 70 genes are tested in 295 tumors (breast
cancer)
 14 metastases/115 px

 Using the criteria from the gene expression profiling, 110 px were
followed for 10yr post-dx
 o They fell into 2 groups: (breast cancer px)
 Good signature: (40% of px) (blue): 96% survival
 Poor signature: (60% of px) (red): 50% survival rate
o Hence predicted cancer progression w. 90% accuracy.
 This data (+ records on efficacy of chemotherapy) indicated that px with a ‘good’
profile drive no benefit from adjuvant chemo
Control of eukaryotic gene expression:
 3. Protein modifications:
o post translational modification of proteins: inactive protein active
protein
o Protein processed
 4. Mutations
 Summary:

Note:
 In eukaryotes, the control of gene expression occurs at many lvls
o The most important is at the level of TRAN
o Other levels include: processing, transport and degradation of mRNA
o At protein level, proteins can be modified, transported and degraded
 Mutations
o DNA damage and repair
 Danage are induced by either replication
errors or external agents
 Unrepaired damage = mutations
 e.g. Cancers, mental, Developmental
anomalies, ageing
o REPLICATION ERRORS (polymerase errors)
repaired by:
 Exonuclease activity of eDNA polymerase
 Mismatch repair system
o DAMAGE caused by external agents – repaired by
 Enzymes specific for the damage (base damage e.g. UV) eg. Glycosylases or
endonucleases
 Excision repair (bulky lesions)
 Recombination (ds breaks)
o UV DAMAGE:
 Thymine -thymine are covalently bonded together =
forming a thymine dimer in the DNA strand;
 this dimer inhibits DNA replication/dead
 ‘bumps’ in DNA + disrupts H-bonding between bases
 Light repair: enzyme is activated by visible light PRE= uses
blue light to break covalent bonds between T bases, allowing
H bonds to form naturally
 Dark reactivation/excision repair: e.g. UvrABC breaks sugar phosphate backbone
of DNA strand on each side:
 Application: ROLE OF spontaneous somatic mutations in retinoblastoma, =a retinal tumour
o Individuals either present with disease:
 Early in life (as children) usually with both eyes
affected or,
 Late in life with only 1 eye affected
o Caused by changes (mutations) in retinoblastoma 1
(RB1) gene
 Mutation causes retinoblast cells to grow out of
control
 2 copies of RB1 gene in evert cll
o Both DNA needs to be expressed to give rise to tumour
o Hereditary: occurs when 1 DNA is already affected.
Only 1 somatic mutation needed for both pair affected
o Sporadic (non-hereditary): mutations occurs 2x = only
develops retinoblastoma in 1 eye + does not pass
mutation on to future children
 RB1 gene = tumor suppressor gene = regulates cell growth /division; makes the protein (pRb)
– helps stop cells from gowing too quickly
o As long s retinal cell has at least 1 RB1 gene = it will not
form retinoblastoma
o Rb1 gene composition: contains 27 exons and 26 introns.

 MOST GENES COME IN PAIRS – 1 from each parent

o In Sporadic cancer: many mutations can build up in cell
over time, eventually leading to cancer
o With hereditary cancer first mutation is inherited and already present at birth
o People with inherited mutations are born with 1 already damaged gene in all cells of
their body
genetic mutations: inheritance (hereditary)
Yes, you can inherit germline genetic mutations, while somatic mutations occur with no previous
history of the mutation in your family. There are several patterns that genetic mutations can pass
from the parent to a child (hereditary), like:
 Autosomal dominant: Only one parent needs to pass the genetic mutation onto their
child for their child to inherit the mutation. Marfan syndrome is an example of a condition
inherited in this pattern.
 Autosomal recessive: Both parents need to pass the same genetic mutation onto their
child for their child to inherit the mutation. Sickle cell disease is an example of a condition
inherited in this pattern.
 X-linked dominant: Babies assigned male or female have an X chromosome. Only one
mutation on the X chromosome needs to pass from one parent to the child for the child to
inherit the mutation. Fragile X syndrome is an example of a disorder inherited in this
pattern.
 X-linked recessive: If only dad has the mutation, there’s 100% that female offspring will
be carriers and no male offspring will be affected. If only mom had the mutation, there’s a
50% chance that female offspring will be carriers and a 50% chance male offspring will
have the condition. If both parents have the mutation, 50% of male offspring will have the
condition and 100% of female offspring will have the mutation. Color blindness is an
example of a condition inherited in this pattern.
 X-linked: Babies assigned male or female have an X chromosome. Mutations on the X
chromosome can pass in a dominant or recessive pattern, but not every pattern is clear on
how the child acquired the mutation from their parents. Thrombocytopenia is an example
of a condition inherited in this pattern.
 Y-linked: Only babies assigned male at birth have a Y chromosome and can inherit this
type. Only one mutation on the Y chromosome needs to pass to the child to inherit the
mutation. Webbed toes are an example of a condition inherited in this pattern.
 Codominant: Each gene has two parts (one from the egg and one from the sperm). They
usually work together to create a single trait. But sometimes, they each work separately to
produce variations of the trait. Alpha-1 antitrypsin deficiency is an example of a condition
inherited in this pattern.
 Mitochondrial: The mitochondria are the part of a cell that creates energy. Only
mitochondria from the egg survive fertilization, when the two cells come together. So, all
maternal DNA in the embryo come from the egg. This is why mitochondrial inheritance is
also known as maternal inheritance. Leber hereditary optic neuropathy (sudden vision
loss) is an example of a condition inherited in this pattern.
Conditions and Disorders
Outline:
- define gene therapy
- describe which features of condition make them appropriate targets for tx with gene therapy
-outline how gene therapy is delivered
List the risk of gene therapy/difficulties inobtaining successful gene therapy

Gene therapy is the use of genetic material to correct diseases caused by gene defects
 preclinical testing and clinical trials began in late 1980s
 Germ-line therapy – treat sperm or ova – all cells of the individual and subsequent
generations altered – banned
 Somatic cell therapy – treat individuals only – single organ ort tssiesu only ltred current
Monogenic disorders
 = Single gene defect
 Comprise <3% of human diseases
 The pathology is due to the direct loss of protein function
 Tx is transfer of correct gene and correct expression
 Current therapy provide:
o Normal lifespan – 15%
o Reproductive capacity 11%
o Fully socially adapted – 6%

Gene therapy aims to introduce a functional gene to compensate for a malfunctional gene
 introducing a normal CTRF gene into lung cells of a CF sufferers, with a sufficient gene
expression level, may eliminate one of the life-threatening SX of disease

Gene therapy alters expression of specific genes in a patient

 delivery of a functional gene
 Delivery of genes encoding for proteins use to treat an acquired disease
 down regulate gene expression

Multifactorial disorder
e.g. coronary heart disease, DM
- involve several genes PLUS environmental factors
- extensive knowledge of a pathophysiology must be
obtained to develop a gene therapy strategy
- the goal is to achieve a better outcome by maniputing the
phenotype
- manipulating the defective genes may or may not be part
of the strategy

Gene delivery:

Gene therapy: requires a vector to introduce nucleic acid into

target cells
- Viruses
- Plasmids
- Delivery in vivo or ex vivo
- Means to enhance efficient uptake the DNA
- Antisense RNA
- Small molecules that regulate gene expression
- Oligonucleotides resistant to enzyme
degradation

Gene delivery systems – pros and cons

- Virus:
o = Most efficient system
 o Can elicit immune response
o Random insertion
o Size of gene carried can be limited
o Can produce permannt correction
- Liposomes
o Can deliver large amts of DNA
o Random insertion
- NAKED DNA
o Easiest methods
o Limited cell types take up DNA
Vectors
Cautionary tale:
 October 1999 – death from gene therapy
 OTC deficiency died 4 hr after gene therapy
 In vivo approach introduced E1 deleted aAdenovirus with
OTC gene into patients through catheter into the portal
vein
o Used 3.8x10^13 virus particles
o The unusually high no of virus particles elicited an
immune response
o Within 4hrs = fever + blood clots developed

Gene therapy for ADA

- bacterium carrying plasmids with cloned normal human ADA gene T cells with disabled
ADA gene isolated from px
Single gene therapy
- best candidates are where a small mt of gene product will have a big effect and an
excessive will not be deleterious
o ADA: lack of adenosine
o Isolate Tlymphaocutes and transfect wthgene in retrovirus + return to PX
o Repeat every 6-8weeks
o Prosmiing: 10^!! Cell treated - immune reconstition /dec in infections
o Gene corrected eclls increased 25-35% of T cell population
- The down side:
o 14 children in gene therapy trial (IL-2) not produced
o All successfully treated with retroviral vector carrying correct copy of gene
o In response to infection, 1 boy began to synthease WBC appropriately. However, he
developed leukemia
 It was found to be caused by insertion of therapyeitv gene upstream of an
oncogene (Lmo2)
 2003 = FdA replaced a halt on gen therapy trails – using retroviral vectors
inblood steam cells
- No of trials approved worldwide: ~100/yr for past 10 years
- What factors have kept gene therapy from becoming an effective TX for disease?
o Short-lived nature of gene therapy
o Immune response -redu ce effectiveness
o Problems with viral vectors – toxicity, immune and inflammatory responses
o Multigene disorders – most commonly occurring disorders, esp
difficult to tx.
- OCULAR: Targeting cell and expression of gene
o Canine retina injected with AAV2/5 carrying promoter and GFP
gene
 (restores retinal functionsin dogs)
  Cone arresting immunobolanting - only about 60% of photoreceptors
expressed GFP
 Reintal histopathology
 GFP expression in scAAV2/5 and ssAAV2/5
injected eyes (dog 1). A: Pre-INJ shows the fundus
appearance before subretinal injection. post-INJ images
were taken immediately after subretinal injection and
show the resulting retinal detachment
 Prevents photoreceptor degeneration in RPE65-deficeint
dogs
 Retinal ganglion cell – in theiner plexiform laye
o Red antibody – IDENTIFIES CONES ABELS PHOTORECEPTOR
CELLS, COMRINIGN GFP gene targets the rods
o Blue labell = retina cells
- LEBER’S congenital amaurosis:
o = inherited degenerative condition, present from birth
 Extent of vision loss varies, can be quite severe ; a baby may be born with v
poor vision or maybe totally blind (VIISON AU)
o Key protein in visual cycle – encoded by RPE65 gene =in RPE = non functional
 = causes a loss of 11-cis retinal
 Children appear to have an alternative pathways for 11-cis-retinal = that
diminishes with age
 Clinical therapy trial for treating this condition = commenced in 2008 in
UK/USA
 GFP = green fl protein= is a protein produced by jellyfish , emitting
biolyminsence in green zone = gene has been cloned and is used in
molecular biology as a marker illuminate growing cancer tumors, show
development of Alzheimer’s siedase
- Gene doping = is the manipulation of gene expression inspot
o = induction of muscle hypertrophy
o Inc. o2 delivery
o Dec. myotatic lvls
o Alterating mycle phenotype to favour endurance
- Gene therapy = raises ethical issues
o What is considered normal
o Preservation of germ line
o Access and expense

- Gene therapy for COLOUR VISION DEFICIENCY

o Gene therapy used to treat squirrel monkeys that had CV
defiicnecy
- RNA Interference
o RNA interference (RNAi)
 = a method to repress expression of genes at the
transcription lvl by destroying mRNA
 RNAi = discovered when scientist aimed to produce
petunia flowers with darker purple pigment
 Introduction of additional copy of gene, under control fo a
strong pooter, resulted in variegated or white flowers
o Experiments:
 Fire and mellow conducted experiments on a muscle
protein in C. elegans= showed when dsRNA was
added, worms twitch like those containing a mutant
copy of gene
  In 2006, Fire and Mellow was awarded the Nobel prize for physiology for
medicine for discovery
o Significance of discovery:
 Protection from viruses and mobile genetics elements
 Regulation of development
 Interaction with chromatin to mediate expression
- Principle of RNAi (in biotechnology):
o E.G. in AMD – BV GROW AND LEAK INTO AC AREA AND INTERFERE WITH VISION.
 Early clinical trials involved direct injection of dsRNA (targeting VEGF mRNA)
into eyes of participants
o Clinical trails were encouraging = px improved visual acuity
 2007: opoko health launched a phase 2 tails of Bevasiranib (the RNAi
therapeutic)
 2009: the trial terminated because participants vision did not improve
significantly compared to other treatment
o DMO
 Phase II trials for another RNAi therapeutic
for Diabetic Mac degeneration targeting
apoptotic stresss response gene in the
choroid = similarly showed improvement in vision acuity, and no remarkable
safety events
 However this study ending in 2010 showed no superioriy over an existing
available treatment
- RNAi therapeutic challenges:
o Delivery- esp. when required. Systemically
o Endonucleases (expensive carrier being developed)
o Access to body sites – such as panrease or bone marrow
o Safety

dsRNA = is cri
-