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Transcript of genetics-131029144303-phpapp01.pdf
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MOLECULAR CELL
BIOLOGY & GENETIC
DISORDERS
THE CELL
Highly organized structure consist of various
organelles held by the cytoskeleton w radiates from
nuclear membrane to cell plasma membrane
The plasma cell membrane is bilayer of phospholipids
Polar hydrophilic head e.g. phosphatidyl choline
form bilayers (as complete circular structures)
effective barrier impermeable to most H2O-
soluble molecules
Non-polar (insoluble) lipid hydrophobic tail
(commonly 2 long-chain FA)
//
The Cell Membrane
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CELL DYNAMICS Old cellular ptn mopped up by small cofactor molecule
(ubiquitin)
Small 8.5 kDa regulating ptn
Present universally in all living cells
Interacts e these worn ptn via their exposed
hydrophobic residues
A complex containing >5 ubiquitin molecules is
rapidly degraded by large proteolytic multienzyme
26S proteosome
Plays role in regulation of receptor tyrosine kinase
in cell cycle & repair of DNA damage
Failure to remove worn proteins chronic debilitating
disorders e.g. Alzheimer & frontotemporal dementias
(accumulation of ubiquinated ptn w are resistant to
ubiquitin-mediated proteolysis)
Resistant ubiquinated proteins inclusion bodies
found in myositis & myopathies causes
Point mutation in target ptn itself e.g. mutant p53
in cancer
External factor altering normal ptn conformation
proteolytic-resistant shape e.g. CJD
Free radicals It is any atom or molecule w contains 1 or more
unpaired electrons more reactive than the native
species
It is implicated in large number of human diseases
When free radical reacts e non-radical chain
reaction direct tissue damage by membrane lipid
peroxidation
The major free radical species produced in human
body
1) Hydroxyl radical (OH)
The most reactive but others can generate
more reactive species as breakdown products
Can cause genetic mutations by attacking
purines & pyrimidines
2) Superoxide radical (O2-)
Superoxide dismutases (SOD) convert
superoxide to hydrogen peroxide (protective
antioxidant mechanism)
Pt e dominant familial forms of amyotrophic
lateral sclerosis (MND) mutations in gene for
CuZn SOD-1 catalases
Glutathione peroxidases enzymes remove
hydrogen peroxide & generated by SOD in cell
cytosol & mitochondria
3) Nitric oxide (NO)
Alpha-tocopherol, urate, ascorbate & glutathione
remove free radicals by reacting directly & non-
catalytically -tocopherol ( vitamin E)
neurodegeneration
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The principal dietary antioxidants are vitamin E,
vitamin C, -carotene & flavanoids
Heat shock proteins The heat shock response is response to tissue stress
(heat, cytotoxic chemicals & free radicals) mediated
by activation of specific genes specific heat shock
proteins (HSPs)
Functions of HSPs
Transport of ptn in & out of specific cell organelles
Degradation of ptn (often by ubiquitination
pathways)
The unifying feature that activate HSPs
accumulation of damaged IC ptn
HSPs are expressed in a wide range of human cancers
& implicated in tumour cell proliferation,
differentiation, invasion, metastasis, cell death &
immune response
PHAGOCYTOSIS, PINOCYTOSIS & EXOCYTOSIS Phagocytosis
Specialized cells e.g. macrophages & neutrophils
Lysosomes rapidly fuse e phagosomes equally rapid
digestion of contents & recycling
Only triggered when specific cell surface receptors
(macrophage Fc receptor) occupied by their ligand
Pinocytosis Much smaller-scale model of phagocytosis
Continually occurring in all cells
In contrast to phagocytosis receptors for smaller
molecular complexes e.g. LDL surface clumping &
internal accumulation of a protein called clathrin
Clathrin-coated pits pinch inwards as clathrin-coated
vesicles
Clathrin prevents fusion of lysosomes (removal
lysosomal fusion & degradation)
Exocytosis Maintenance of clathrin coat transcellular transit
of contents & their exocytosis at another side of
plasma membrane i.e. apical to basal transcytosis
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Some of these vesicles rapidly fuse e plasma
membrane & exocytose their contents
Other vesicles do not immediately fuse e plasma
membrane
The clathrin-coated vesicles have additional lipid
bilayer embedded proteins called v-SNAREs (signal &
response elements) interact e target organelle
membrane proteins called t-SNAREs vesicle fusion
is therefore specific in the correct place & in the
correct time e.g. neuronal transmitter vesicles
MEMBRANE TRANSPORT & ION CHANNELS Plasma membrane is freely permeable to
Gases e.g. O2, CO2 and N2
Small uncharged molecules e.g. H2O (not H+ & OH)
& urea
Larger hydrophobic lipid-soluble molecules e.g.
steroids
Large uncharged molecules (G, aa & nucleotides) and
small charged ions (K, Na, Ca, Cl, Mg & HCO3) cannot
pass unless via specific transport ptn embedded in
plasma membrane
2 Structural types of transport molecules/complex
1) Channel proteins
Open a channel in the lipid membrane
Allow specific solute to pass through
2) Carrier proteins
Slower in action
Shuttling the solute across
Facilitating diffusion down a gradient across the
membrane OR actively pumping solutes against
the gradient using ATP as energy
RECEPTORS Membrane surface receptors pass their EC signal
across plasma membrane to cytoplasmic 2ry signalling
molecules
Membrane-bound receptors is subclassified according
to mechanism by which they activate signalling
molecules
Ion channel linked
G-protein linked
Enzyme linked
Structure of plasma membrane receptors
Serpentine 7 transmembrane domains e.g. LH
receptor
Transmembrane with large EC & IC domains e.g.
EGF receptor
Transmembrane with large EC domain only e.g.
macrophage scavenging receptors
Entirely linked to outer membrane leaflet by lipid
moiety known as GPI anchor (glycan phosphatidyl
inositol) e.g. T cell receptor
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Function of membrane receptors is to initiate 2ry
message activation of specific enzyme or DNA-
binding protein. This may involve
G-protein-linked receptors Once activated by ligand binds trimeric complex (,
, ) anchored to inner surface of plasma membrane
The complex is GTP-binding protein or G-protein then
interacts e enzyme complexes anchored to inner
leaflet of the membrane
These complexes 1 or all 3 of 2ry messengers
cyclic AMP (cAMP) Ca2+ ions
Inositol trisphosphate / diacylglycerol (IP3/DAG)
Enzyme-linked surface receptors These receptors usually have single transmembrane
spanning region & cytoplasmic domain e intrinsic
enzyme activity OR bind & activate other membrane
bound or cytoplasmic enzyme complexes
4 classes of enzymes have been designated
1) Guanylyl cyclase-linked receptors
e.g. ANP receptor w produce cGMP
In turn activates cGMP-dependent kinase (G-
kinase) binds to & phosphorylates serine &
threonine residues of specific 2ry messengers
2) Tyrosine kinase receptors
e.g. PDGF receptor
Specifically phosphorylate kinases on small set
of IC signalling proteins OR associate e ptn e
tyrosine kinase activity
3) Tyrosine phosphatase receptors
e.g. CD45
Remove phosphates from tyrosine residues of
specific IC signalling proteins
4) Serine/threonine kinase receptors
e.g. TGF- receptor
Phosphorylate specific serine & threonine
residues of IC signalling proteins
Many IC receptors that bind lipid-soluble ligands e.g.
steroid hormones (Pg, cortisol), T3/T4 often change
shape in response to binding their ligands enter the
nucleus & interact directly e specific DNA sequences
The fluid component inside the cell membrane
It contains many specialized organelles
Endoplasmic reticulum (ER) Consists of interconnecting tubules or flattened sacs
(cisternae) of lipid bilayer membrane
Cytoplasm
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It may contain ribosomes on the surface (rough
endoplasmic reticulum RER) & when absent (smooth
endoplasmic reticulum SER)
ER is involved in processing of ptn ribosomes
translate mRNA to 1ry sequence of aa of ptn peptide
chain
This chain is synthesized in the ER where it is folded
& modified into mature peptides
ER is the major site of drug metabolism
Golgi apparatus Consist of flattened cisternae similar to ER
Characterized as stack of cisternae from w vesicles
bud off from the thickened ends
The 1ry processed peptides of ER are exported to
Golgi apparatus for maturation into functional ptn e.g.
glycosylation of ptn to be excreted before packaging
into secretory granules & cellular vesicles that bud off
the ends
Lysosomes Dense cellular vesicles contain acidic digestive
enzymes
Fuse e phagocytotic vesicles from outer cell
membrane digest contents into small biomolecules
capable of cross lysosomal lipid bilayer to cytoplasm
Lysosomal enzymes can be released outside cell by
fusion of the lysosome e plasma membrane
Lysosomal action is crucial to function of macrophages
& PMNs in killing & digesting infective agents, tissue
remodelling during development & osteoclast
remodelling of bone
Peroxisomes Dense cellular vesicles contain enzymes catalyse the
breakdown of H2O2
They are involved in metabolism of bile & FA
Primarily concerned e detoxification e.g. d-amino acid
oxidase & H2O2 catalase
The inability to function rare metabolic disorders
e.g. Zellwegers syndrome & rhizomelic dwarfism
Mitochondria The powerhouse of the cell
Each mitochondrion has 2 lipid bilayer membranes
The outer membrane
It contain many gated receptors import raw
materials like pyruvate & ADP oxaloacetate &
ATP
Proteins of Bcl-2/Bax family are incorporated in
the outer membrane can release mitochondrial
enzymes that trigger apoptosis
The inner membrane
Highly infolded to form cristae toits effective
surface area
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Contains transmembrane enzyme complexes of
electron transport chain generate H+ ion
gradient drives adjacent transmembrane ATPase
complex to form ATP from ADP & Pi
The inner matrix
It possesses several copies of its own DNA in
circular genome
It contains enzymes of Krebs cycle that generate
substrates of both electron transport chain
(FADH2 & NADH) & central metabolism e.g.
succinyl CoA, -oxoglutarate, oxaloacetate
2ry messengers are molecules that transduce a signal
from a bound receptor to its site of action
There are essentially 4 mechanisms by which 2ry
messengers act (cross talk & rarely activated alone)
cAMP IP3/DAG
Ca2+ ions Protein phosphorylation
cAMP, IP3/DAG & Ca2+ ions Generation of cAMP by G-protein-linked receptors
cellular cAMP bind & activate specific cAMP
binding proteins dimerize & enter nucleus
interact e set DNA sequences (cAMP response
elements)
Cofactors in cAMP response element binding
proteins (CREB) are co-activated & interact e
phosphorylation pathway
Other G-protein complexes activate inner
membrane bound phospholipase complexes cleave
membrane phospholipid-polyphosphoinositide (PIP2)
1) Inositol trisphosphate (IP3) H2O soluble
molecule floats in cytoplasm interacts e gated
ion channels in ER (or sarcoplasmic reticulum in
muscle cells) rapid release of Ca2+
2) Diacylglycerol (DAG) lipid soluble that remains at
membrane activates a serine/threonine kinase
protein kinase C
The cellular calcium-binding proteins & ion pumps
rapidly remove Ca2+ from cytoplasm back into storage
compartment e.g. ER
Free Ca2+ interacts e target proteins in cytoplasm
phosphorylation / dephosphorylation cascade
activated DNAbinding proteins entering nucleus
Protein phosphorylation The principal route for ptn phosphorylation cascades
is from dimerization of surface ptn kinase receptors
Tyrosine kinase receptors phosphorylate each other
when ligand binding brings IC receptor components
into close proximity
Secondary Messenger
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Inner membrane & cytoplasmic targets of these
activated receptor complexes are ras, ptn kinase C &
ultimately MAP (mitogen activated ptn) kinase, Janus-
Stat pathways or phosphorylation of IB release its
DNA-binding protein, nuclear factor kappa B (NFB)
IC signalling proteins usually contain conserved non-
catalytic regions called SH2 & SH3 (SRC homology
regions 2 & 3) SH2 region binds to phosphorylated
tyrosine & SH3 domain is implicated in recruitment of
intermediates that activate ras proteins
Like G-proteins ras (& its homologous family
members rho / rac) switch between inactive GDP-
binding state & active GTP-binding state
NFB conformational change & enter nucleus
initiates transcription of specific genes
Lipid-soluble ligands e.g. steroids not need 2ry
messengers cytoplasmic receptors once activated
enter nucleus as DBP alter gene expression directly
Complex network of structural ptns w regulates
Shape of the cell
Cell ability to traffic internal cell organelles &
move in response to external stimuli
The major components
1) Microtubules
Made of 2 ptn subunits & tubulin (50 kDa)
Continuously change length highway
transporting organelles through cytoplasm
2 motor microtubule associated ptns (dynein &
kinesin) antegrade & retrograde movement
(dynein also beating of cilia)
During interphase microtubules rearranged
by microtubule organizing centre (MTOC) w
consists of centrosomes containing tubulin &
provide structure on w daughter Chr can
separate
The Cytoskeleton
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Another ptn involved in binding of organelles to
microtubules cytoplasmic linker protein
(CLIP)
Drugs disrupt microtubule assembly (colchicine
& vinblastine) affect positioning & organelles
morphology
Anticancer drug paclitaxel causes cell death
by binding to microtubules & stabilizing them
organelles cannot move mitotic spindles not
formed
2) Intermediate filaments
Form network around nucleus & extend to cell
periphery
They make cell-to-cell contacts e adjacent cells
via desmosomes
They make contact e basement matrix via
hemidesmosomes
Function structural integrity (prominent in
cellular tissues under stress)
Intermediate filament fibre ptns are specific to
embryonic lineage of the cell e.g. keratin
intermediate fibres only found in epithelial cells
3) Microfilaments
Muscle cells contain
o Actin highly ordered structure of actin
(globular ptn, 4244 kDa)
o Myosin filaments form contractile system
These filaments also present in nonmuscle cells
as truncated myosins (e.g. myosin 1), in cytosol
(forming contractile actomyosin gel) & beneath
plasma membrane
Cell movement is mediated by anchorage of
actin filaments to plasma membrane at adherent
junctions between cells non stressed
coordination of contraction between adjacent
cells of tissue (similarly, vertical contraction of
tissues is anchored across cell membrane to
basement matrix at focal adhesion junctions
where actin fibres converge)
Actinbinding ptns e.g. fimbria modulate
behaviour of microfilaments & their effects are
often Ca dependent
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Actin-associated ptns can be tissue type
specific e.g. actin-binding troponin is complex
of 3 subunits & 2 of these have isomers w are
only found in cardiac muscle
Alterations in cells actin architecture are controlled
by activation of small ras-like GTP-binding proteins
rho & rac involved in rearrangement of cell during
division dysfunctions of these ptns are associated e
malignancy
EC domains form junctions between cells to form
tissues
Types of junction between cells
1) Tight junctions (zonula occludens)
Situated at ends of margins adjacent to
epithelial cells e.g. intestinal & renal cells
Form barrier to movement of ions & solutes
across the epithelium (may be variably leaky to
certain solutes)
The ptns responsible for intercellular tight
junction closure (claudins) selective
expression ein tissue & regulate w ions pass
Mutations of claudin-16 (expressed in thick
ascending loop of Henle where Mg is
reabsorbed) abnormal Mg reabsorption of
Gitelmans syndrome
2) Adherent junctions (zonula adherens)
Continuous on basal side of cells
Contain cadherins
The major site of attachment of IC
microfilaments
Intermediate filaments attach to desmosomes
areas of thickened membranes of 2 adjacent
cells
Hemidesmosomes attach cells to basal lamina &
also connected to intermediate filaments
Transmembrane integrins link EC matrix to
microfilaments at focal areas where cells also
attach to their basal laminae
In blistering skin disorders auto-Ab damage
by attacking tight junction desmosomal proteins
e.g. desmoglein-3 in pemphigus vulgaris &
desmoglein-1 in pemphigus foliaceus
3) Gap junctions
Allow substances to pass directly between cells
eout entering ECF
Ptn channels (connexins) are lined up between 2
adjacent cells & allow solutes passage up to MW
1000 kDa e.g. aa, sugars, ions, messengers
Channels diameter is regulated by IC Ca2+, pH &
voltage
Intercellular Connections
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Connexins 6 subunits surrounding channel &
their isoforms in tissues are encoded by
different genes
Mutant connexins disorders e.g. X-linked
form of CharcotMarieTooth disease
Major families of cell adhesion molecules
1) Cadherins
Cadherins establish molecular links between
adjacent cells
They form zipper-like structures at adherens
junctions
Through these junctions, bundles of actin
filaments run from cell to cell.
Related molecules e.g. desmogleins form the
main constituents of desmosomes (anchoring
sites for intermediate filaments)
The expression of specific adhesion molecules in
the embryo is crucial for cell migration &
differentiation of tissues
2) Integrins
They are membrane glycoproteins e &
subunits w exist in active & inactive forms
They principally bind to EC matrix components
e.g. fibrinogen, elastase & laminin
The aa sequence arginineglycineaspartic acid
(RGD) potent recognition sequence for
integrin binding
Integrins replace cadherins in focal membrane
anchorage of hemidesmosomes & focal adhesion
junctions
The active form of integrin can come as result
of cytoplasmic signal that causes conformational
change in EC domain affinity for its ligand
o The inside-out signalling occurs when
leucocytes are stimulated by bacterial
peptides leucocyte integrin affinity for
Ig super families structures e.g. Fc portion
of Ig immunoglobulin
o The outside-in signalling follows binding of
ligand to integrin & stimulate 2ry signals
diverse events e.g endocytosis, proliferation
& apoptosis
Defective integrins are associated e many
immunological & clotting disorders e.g. Bernard
Soulier syndrome & Glanzmanns thrombasthenia
3) Ig superfamily cell adhesion molecules (CAMs)
Ig-like structures domains
Neural cell adhesion molecule (N-CAM)
o Predominantly in nervous system
o Mediates homophilic adhesion
Cell Adhesion & Molecules
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o When bound to identical molecule on another
cell N-CAM associate laterally with
fibroblast growth factor receptor
stimulate tyrosine kinase activity of that
receptor growth of neurites (Adhesion
molecules can trigger cellular responses by
indirect activation of other types of
receptors)
o The placenta and gastrointestinal
Placenta & GIT also express Ig superfamily
members but unclear function
4) Selectins
Selectins interact e CHO ligands or mucin
complexes on leucocytes & endothelial cells
(most adhesion molecules bind to other ptn)
L-selectin (CD62L) is found on leucocytes
homing of lymphocytes to lymph nodes
E-selectin (CD62E) appears on endothelial cells
after activation by inflammatory cytokines
small basal amount of E-selectin in many
vascular beds is necessary for leucocytes
migration
P-selectin (CD26P) stored in granules of
platelets & WeibelPalade bodies of endothelial
cells it moves plasma membrane upon
stimulation of these cells
All 3 selectins play part in leucocyte rolling
A nucleus is present in all eukaryotic cells that divide
Contains human genome & bound by 2 bilayer lipid
membranes, the outer is continuous e ER
Nuclear pores present in membranes allow passage
of nucleotides & DNA interacting ptns in AND mRNA
out
The genome consists of DNA plus all apparatus for
replication & transcription into RNA
The Nucleus & its responses
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Types of cell division
Meiosis
Occurs in germ cells only
Chromosome complement is halved (haploid) & at
fertilization the union of 2 cells restores full
complement of 46 chromosomes
Mitosis
Occurs in dividing cells after fertilization
Results in 2 identical daughter cells
Chromosomes are only visible during cell division
A nucleolus is dense area ein the nucleus rich in
ptns & RNA synthesis of rRNA & ribosomes
THE CELL CYCLE Cells in quiescent G0 phase (G, gap) of the cycle are
stimulated by receptor-mediated actions of growth
factors e.g. EGF, PDGF, IGF via IC 2nd messengers
Stimuli are transmitted to nucleus activate
transcription factors initiation of DNA synthesis
then mitosis & cell division
Cell cycling is modified by cyclin family of ptns
Cyclin & cyclin-dependent kinases Coordinated cyclic expression of cyclin-dependent
kinases (Cdk) drives cell replication cycle
Cell cycle is catalysed by Cdk w are activated by class
of ptns called cyclins (Cyc)
After stimulation from pro-mitotic EC signal e.g.
growth factor G1 cyclinCdk complexes (CycB
/Cdk4/6; CycE/Cdk2) become active to prepare cell
for S phase expression of transcription factors
expression of S cyclins (CycB/Cdk2) & enzymes
required for DNA replication
G1 cyclinCdk complexes degradation of molecules
that function as S phase inhibitors by targeting them
for ubiquitination
Active S cyclinCdk complexes phosphorylate ptns
that make up pre-replication complexes assembled
during G1 phase on DNA replication origins serves 2
purposes
1) Activate each already assembled pre-replication
complex
2) Prevent new complexes from forming
This ensures that every portion of genome will be
replicated once only
Mitotic cyclinCdk complexes e.g. CycB/CdK2
(synthesized but inactivated during G2 phase)
initiation of mitosis by stimulating downstream ptns
involved in chromosome condensation & mitotic spindle
assembly
Apoptosis (programmed cell death) Deliberate activation of constituent genes responsible
for their own demise
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Necrotic cell death
External factor e.g. hypoxia, toxins damages cells
physiology cell disintegration
Influx of water & ions cellular organelles swell
rupture
Cell lysis release of lysosomal enzymes in EC
environment acute inflammatory responses in
vivo
Apoptotic cell death
Chromatin aggregation + nuclear & cytoplasmic
condensation in distinct membrane bound vesicles
(apoptotic bodies)
Organelles remain intact
Cell blebs intact membrane vesicles
No inflammatory response
Cellular blebs & remains are phagocytosed by
adjacent cells & macrophages
This process requires energy (ATP) and several Ca2+ &
Mg2+ dependent nuclease systems activation cleave
nuclear DNA at the inter-histone residues
Endonuclease destroys DNA following apoptosis this
involve enzyme caspase (cysteine-containing aspartase-
specific protease) w activate CAD (caspaseactivated
DNase)/ICAD (inhibitor of CAD) system destroy
DNA
Regulated apoptosis is essential for
Tissue structure formation in embryogenesis
Wound healing
Normal metabolic processes e.g. autodestruction of
endometrium to cause menstruation
Chemotherapy & radiotherapy only work if they can
trigger tumour cells own apoptotic pathways
Several factors initiate apoptosis but in general there
are 2 signalling pathways
1) The extrinsic pathway
Involved in processes e.g. tissue remodelling &
induction of immune selftolerance
Triggered by death receptors on cell surface e
internal death domain complexes multiply pro-
caspase 8 molecules release of initiator caspase
8 cleaves pro-caspase 3 caspase 3 + other
effector caspases activate DNA cleavage, cell
condensation & fragmentation
Death receptors are members of TNF receptor
superfamily include CD95 (APO-1/Fas), TRAIL
(TNF-related apoptosis ligand)-R1, TRAIL-R2,
TNF-R1, DR3 & DR6
2) The intrinsic pathway
Initiated at the mitochondrial level centres on
release of cytochrome C from mitochondria
Cellular stress (growth factor withdrawal & p53
cell cycle arrest) expression of pro-apoptotic
Bcl-2 family of ptns, Bax & Bak tetrameric
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The Fas protein & Fas ligand (FasL) are 2 ptns that interact to activate apoptotic pathway. Fas & FasL are both members of TNF family Fas is part of transmembrane receptor family & FasL is part of membraneassociated cytokine family. When the homotrimer of FasL binds to Fas, it causes Fas to trimerize & brings together the death domains (DD) on the cytoplasmic tails of ptn. The adaptor protein, FADD (Fas-associating ptn e death domain), binds to these activated death domains & they bind to pro-caspase 8 through a set of death effector domains (DED)
complexes imbed to outer mitochondrial
membrane permissive pores
Cytochrome C released from mitochondria binds
Apaf1 complex called apoptosome activates
initiator caspase (caspase 9) activates effector
caspase (caspase 3)
Other ptns released from damaged mitochondria
(Smac/DIABLO & Omi/HtrA2) counteract
effect of IAPs (inhibitor of apoptosis ptns)
normally bind & prevent activation of pro-caspase 3
Antiapoptotic Bcl-2 ptn, when incorporated as
member of Bak/Bax pore complex mitochondrial
pore non-permissive to release of cytochrome C &
anti-IAPs
There is amplification link between extrinsic &
intrinsic apoptotic pathways caspase 8 cleaves Bcl-2
family member, tBid formation of Bcl-2/Bax/Bak
pore complexes if this complex is predominantly of
pro-apoptotic members of Bcl-2 family
apoptosome/caspase 9 & mitochondrial anti-IAPs
apoptotic activation of effector caspases 3
Conversely, overexpression of antiapoptotic Bcl-2
intrinsic & extrinsic apoptotic signalling
Stem cells The majority of our cells are terminally differentiated
& contain the blueprint to produce all the ptns of the
body but each tissue has permanently deactivated all
except those required for the specialized function of
the cells
Therefore we must have nests of cells ein all
different tissues that have not shut down their
genetic blueprint
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These stem cells give rise to daughter cell
(differentiated & limited ability to replicate) &
daughter cell w will not differentiate & has the
infinite ability to replicate
In mammals source categories of stem cells
Embryonic stem cells derived from blastocysts
Adult stem cells found in adult tissues
Cord blood stem cells found in umbilical cord
The source of stem cells can also be subcategorized
by potency (specifies the potential to differentiate to
different cell types)
Totipotent stem cells
Produced from fusion of egg & sperm cell
Produced by 1st few divisions of fertilized egg
Can differentiate to embryonic &
extraembryonic cell types
Pluripotent stem cells
The descendants of totipotent cells
Can differentiate to cells derived from any of
the 3 germ layers
Multipotent stem cells
Produce only cells of closely related family e.g.
haematopoietic stem cells RBCs, WBCs, etc.
Unipotent cells
Produce only 1 cell type
Have the property of self-renewal (w
distinguishes them from non-stem cells)
MOLECULAR BIOLOGY
Genetic information is stored in form of double-
stranded DNA
Each strand of DNA is made up of deoxyribose
phosphate backbone & series of purine (adenine (A) &
guanine (G)) and pyrimidine (thymine (T) & cytosine
(C)) bases of the nucleic acid
The length of DNA is generally measured in numbers
of base-pairs (bp)
The monomeric unit in DNA (& RNA) is the nucleotide
w is a base joined to sugarphosphate unit
The 2 strands of DNA are held together by hydrogen
bonds between the bases
There are only 4 possible pairs of nucleotides TA,
AT, GC & CG
The 2 strands twist to form double helix e major &
minor grooves
The large stretches of helical DNA are coiled around
histone ptns nucleosomes & further condensed into
chromosomes that are seen at metaphase
DNA Structure & Function
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17
Gene is portion of DNA that contains codes for
polypeptide sequence
3 adjacent nucleotides (codon) code for particular aa
e.g. AGA for arginine
Only 20 common aa but 64 possible codon combinations
make up genetic code most aa encoded by >1 triplet
Other codons used as signals for initiating or
terminating polypeptide-chain synthesis
Genes consist of lengths of DNA that contain
sufficient nucleotide triplets to code for the
appropriate number of aa in polypeptide chains of
particular ptn
Genes vary greatly in size (most extend over 2040
kbp) but few e.g. gene for muscle ptn dystrophin can
extend over millions of bp
In bacteria the coding sequences are continuous but in
higher organisms these coding sequences (exons) are
interrupted by intervening sequences that are non-
coding (introns) at various positions
Some genes code for RNA molecules w will not be
translated to ptns code for functional rRNA &
tRNA)
Micro RNAs single-stranded RNA molecules of
about 22 nucleotides inactivate specific mRNA &
disrupt expression of their ptns regulating cell
proliferation & apoptosis (in turn they are inactivated
by DNA methylation)
Conversion of genetic information to polypeptides &
ptns relies on transcription of sequences of bases in
DNA to mRNA
mRNA
Found mainly in nucleolus & cytoplasm
Polymers of nucleotides containing ribose
phosphate unit attached to base
The bases are A, G, C & uracil (U)
RNA is ss molecule but can hybridize e
complementary sequence ssDNA
Transcription & Translation
Genes
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Genetic information is carried from nucleus to
cytoplasm by mRNA act as template for ptn
synthesis
Each base in mRNA is lined up opposite to
corresponding base in DNA (C-G, G-C, U-A & A-T)
Gene always read in 5-3 orientation & at 5 promoter
sites w specifically bind enzyme RNA polymerase
(indicate where transcription is to commence)
2 AT-rich promoter sites are present in eurokaryotic
genes
1st (TATA box) is located about 25 bp before the
transcription start site
2nd (CAAT box) is 75 bp before the start site
Initial mRNA is complete copy of 1 strand of DNA
contains introns & exons
While still in nucleus mRNA post transcriptional
modification 5 & 3 ends are protected by addition
of inverted guanidine nucleotide (CAP) & chain of
adenine nucleotides (Poly A tail) activity of specific
5 mRNA nucleases is to remove the cap & further
regulated by Poly A tail w must 1st be removed by
other degradation enzymes
In higher organisms 1ry transcript mRNA is further
processed inside nucleus introns spliced out
(splicing by small nuclear RNA in association e specific
ptns)
Alternative splicing is possible whereby entire exon
can be omitted >1 ptn coded from same gene
Processed mRNA migrates out of nucleus to
cytoplasm polysomes (groups of ribosomes) become
attached to mRNA ribosomes consist of subunits
composed of small RNA molecules (rRNA) & ptns
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rRNA components are key to binding & translation of
genetic code held by ribosomes & triplets of adjacent
bases on mRNA called codons are recognized by
complementary sequences or anti codons in tRNA
each tRNA molecule carries aa that is specific to anti
codon
As the ribosome passes along mRNA in the 5-3
direction (zipper linking) aa transferred from tRNA
molecules & linked by ribosome polypeptide chain
1st 20 or more nucleotides are recognition & regulatory
sequences and untranslated but necessary for
translation
Translation begins when triplet AUG (methionine) is
Encountered all ptns start e methionine but it is
often lost as the leading sequence of aa of native
peptides is removed during ptn folding
Similarly Poly A tail is not translated & is preceded by
stop codon UAA, UAG or UGA
Gene expression is controlled at many points in steps
between translation of DNA to ptns
Ptns & RNA molecules are in constant state of
turnover
For many genes, transcriptional control is the key
point of regulation
Deleterious (even oncogenic) changes to cell may arise
through fault in expression of particular gene e.g. over
expression due to non-break down of mRNA
Pathway that stops gene expression by RNA
degradation (RNA interference, RNAi)
Transcriptional control Gene transcription (DNA to mRNA) is not spontaneous
event ? only result of interaction of number of DNA
binding ptns (DBP) e genomic DNA
Regulation of gene expression must 1st start e opening
up of double helix of DNA in the correct region of Chr
in order to do this ptn molecules that recognize
the outside of DNA helix has evolved these DBP
interact e major groove of DNA double helix
bp composition of DNA sequence can change geometry
of DNA helix to facilitate fit of DBP e its target
region e.g. C-G rich areas form Z structure DNA helix,
sequences such as AAAANNN slight bend & if
repeated every 10 nucleotides it produces pronounced
curves
DBP that recognize these distorted helices opening
up (or prevent opening) of the helix so the gene may
be transcribed
The Control of gene expression
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20
Structural classes of DBP 4 basic DBP (according to structural motifs)
Class of DBP Examples
Helixturnhelix CREB (cAMP response element binding ptn) Zinc finger Steroid & thyroid hormone receptors
Retinoic acid & vitamin D receptors Bcl-6 oncogene product (lymphoma) WT1 oncogene product (Wilms tumour) GATA-1 erythrocyte differentiation & Hb expression factor BRCA 1 (familial breast cancer)
Leucine zippers c-jun cell replication oncogene c-fos cell replication oncogene
Helixloophelix myc oncogene mad oncogene max oncogene
Control regions & proteins DBP act as regulators of gene expression in 3
different ways promoters, operators & enhancers
Promoters
RNA polymerases bind to promoter region normally
adjacent to transcribed sequence of DNA
In eukaryotes active transcription is possible only
when number of DBP & DNA associated proteins
come together & interact (general transcription
factors) these ptns thought to assemble at
promoter sites used by RNA polymerases e.g. Pol II
that are characterized by specific motifs e.g.
TATA sequence
Operator
Other DNA regulator ptns operate in close
proximity to site of promoter binding (operator
ptns/regions & act either as repressors by binding
to DNA sequences ein promoter site or as +ve
regulators facilitating RNA polymerase binding
Enhancer
Enhancer sequences are >200 bp away from site of
transcription initiation
Binding of regulator ptns to enhancer regions
(several 100 bases from promoter site)
upregulates the expression
This turns out to be distance favourable for DNA
to loop back on itself eout straining backbone
bonds of DNA double helix
GAL4 enhancer of yeast aid binding of transcription
factors to TATA region of promoter catalyst for
general transcription factor assembly & RNA
polymerase activity
In mammals cAMP response element (CRE) acts to
IC cAMP activation & release of CREB
transcription rate (but may alsotranscription)
Repressors cantranscription of gene by binding to
regulatory sequence & blocking +ve regulators or by
interfering e promoter ptn assembly
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21
CHROMOSOMES, INTRONS & THE SIZE OF HUMAN GENOME
Coiling around histones & structural regions e.g.
centromeres & telomeres requires regions of DNA
devoted specifically to the purpose of packaging
10% of human DNA is highly repetitive (satellite DNA)
long arrays of tandem repeats these regions tend
to be supercoiled around histones in condensed regions
(heterochromatin)
In contrast most other DNA regions are relatively
uncondensed (euchromatin)
The remaining DNA is either moderately repetitive
(30% of genome) or codes for unique genes (gene
families occupying 2% of genome)
PREPARATION OF GENOMIC DNA 1st step in studying DNA of individual involves
preparation of genomic DNA
It is simple procedure in w any cellular tissue including
blood can be used
Cells are lysed in order to open their cell & nuclear
membranes releasing chromosomal DNA
Digestion of all cellular ptn by add of proteolytic
enzymes genomic DNA is isolated by chemical
extraction e phenol
DNA is stable & can be stored for years
RESTRICTION ENZYMES & GEL ELECTROPHORESIS
Restriction enzymes cut dsDNA at specific sites
Whenever human genomic DNA is cut e EcoRI same
restriction fragments (restriction fragment length
polymorphisms, RFLPs) are produced
As DNA is ve charged molecule genomic DNA
fragments can be separated according to their size &
charge by electrophoresis through a gel matrix
DNA migrates to +ve anode & small fragments move
more quickly DNA fragments separate out
Pulsed-field gel electrophoresis (PFGE) can be used to
separate very long pieces of DNA (100s of kilobases)
HYBRIDIZATION TECHNIQUES When 2 strands separated(e.g. by heating) they will
always re stick because of their complementary base
sequences
Therefore presence of particular gene can be
identified using gene probe consisting of DNA or RNA
e base sequence complementary to the sequence of
interest
Tools for Molecular Biology
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22
DNA probe is piece of ssDNA that can be labelled e
radioactive isotope (usually 32P) or fluorescent signal
will locate & bind to its complementary sequence
Hybridization is exploited in number of techniques
including
Southern blot DNA fragments separated by gel
electrophoresis & transferred onto membrane
sheet
Northern blot RNA separated by gel
electrophoresis & transferred onto membrane
sheet
In situ hybridization localization of native
nucleic acid sequences ein the cell & its component
organelles, including chromosomes
THE POLYMERASE CHAIN REACTION (PCR) Minute amounts of DNA can be amplified over million
times ein few hours
The technique has 3 steps
ds genomic DNA is denatured by heat into ssDNA
Then cooled to favour DNA annealing & primers
bind to their target DNA
Finally DNA polymerase extend the primers in
opposite directions using target DNA as template
After one cycle 2 copies of dsDNA, after 2 cycles
4 copies
Real-time PCR (RT-PCR) Also called quantitative real time PCR (QRT-PCR)
Simultaneous quantification & amplification of given
DNA sequence
It can be used to determine whether specific
sequence is present in sample e.g. viral genome & if
present, the number of copies in the sample
RT-PCR is combined e reverse transcription PCR to
quantify low abundance mRNA enabling researcher to
quantify relative gene expression at particular time in
particular cell/tissue
Expression microarrays/gene chips It is methodology developed to examine relative
abundance of mRNA for 1000s of genes present in
cells/tissue of different types e.g. to examine changes
in gene expression from normal tissue to that of
malignant colonic polyps
The basic technology is the ability to immobilize
sequences of DNA complementary to specific genes or
different regions of known genes onto solid surface in
precise microdot arrays
Total mRNA extracted from one tissue & labelled e
fluorescent tag Cy3-green & mRNA from 2nd tissue e
fluorescent tag Cy5-red The 2 fluorescent tagged
total mRNA samples mixed in 1 : 1 ratio & washed over
DNA gene chips mRNA for specific genes will bind
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23
to their complementary microdot & detected by laser-
induced excitation of fluorescent tag position, light
wavelength & intensity recorded by scanning confocal
microscope relative intensity of Cy5-red : Cy3-
green is reliable measure of relative abundance of
specific mRNAs in each sample
Yellow equal binding of both fluorescent tagged
mRNA
Black no hybridization
Red overexpression
Green under expression
Power of the system many 1000s of genes screened
for expression & relative expression in normal &
diseased tissue
DNA CLONING Particular DNA fragment of interest isolated &
inserted to genome of simple self replicating organism
or organelles e.g. viruses & plasmids
Vectors include bacteriophage viruses; plasmids
Each vector takes optimum size of cloned DNA insert
(viruses accommodate only small sequences, larger
fragments can be inserted in plasmid & larger in yast
Chr)
Hybrid between plasmid & bacteriophage (cosmid)
constructed artificially & has ability to clone
reasonably large sequences as plasmids ein host
bacteria trick bacteriophages in packaging them to
viral body & this viral body is then able to infect
target bacteria efficient transfection rates
DNA fragment of interest is inserted in the vector
DNA sequence using enzyme ligase (in vitro) cloning
& creates many copies of recombinant DNA molecule
(in vivo)
Alternatively it could be cDNA w has been copied
from mRNA sequence by reverse transcriptase enzyme
ssDNA DNA polymerase dsDNA contains all
sequences necessary for functional gene but unlike
genomic DNA it lacks introns
HUMAN CHROMOSOMES Each diploid cell nucleus contain 6109 DNA bp in Chr
Chromosomes contain one linear molecule of DNA
wounded around histone in small units (nucleosomes)
Diploid human cells have 46 chromosomes (23
inherited from each parent) 23 homologous pairs
22 pairs of autosomes + 2 sex chromosomes(XY/XX)
Chromosomes classified according to their size &
shape (the largest is Chr 1)
The Biology of Chromosomes
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24
The constriction in Chr is centromere metacentric
(in middle of Chr) or acrocentric (at one extreme end)
Centromere divides Chr into short arm (p) & long arm
(q) e.g. CFTR gene (of cystic fibrosis) maps to 7q21
on Chr 7 in long arm in band 21
Indications for chromosomal analysis
Antenatal
Pregnancies in women >35 years
+ve maternal serum screening for aneuploid
pregnancy
U/S features consistent e aneuploid fetus
Severe fetal growth retardation
Sexing of fetus in X-linked disorders
In the neonate
Congenital malformations
Suspicion of trisomy or monosomy
Ambiguous genitalia
In the adolescent
1ry amenorrhoea or puberty development failure
Growth retardation
In the adult
Screening parents of child e chromosomal
abnormality for further genetic counselling
Infertility or recurrent miscarriages
Learning difficulties
Certain malignant disorders e.g. leukaemias &
Wilms tumour
THE X CHROMOSOME & INACTIVATION 1 of 2 X Chr in cells of becomes transcriptionally
inactive cell has only 1 dose of X-linked genes (X
inactivation or Lyonization phenomenon)
Inactivation is random & can affect either X
chromosome
TELOMERES & IMMORTALITY Ends of Chr (telomeres) do not contain genes but many
repeats of hexameric sequence TTAGGG
Replication of linear Chr start at coding sites (origins
of replication) ein main body of Chr (not at 2 extreme
ends)
Extreme ends are susceptible to ssDNA degradation
back to dsDNA cellular ageing measured as genetic
consequence of multiple rounds of replication e
consequential telomere shortening Chr instability &
cell death
Stem cells have longer telomeres > daughter
Germ cells replicate eout shortening of their
telomeres because they express enzyme telomerase
(protects against telomere shortening by acting as
template primer at extreme ends of Chr)
Most somatic cells (unlike germ & embryonic cells)
switch off activity of telomerase after birth
Many cancer cells reactivate telomerase contributing
to their immortality
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25
THE MITOCHONDRIAL CHROMOSOME In addition to 23 pairs of Chr in nucleus, mitochondria
in cytoplasm have their own genome
Mitochondrial Chr is circular DNA (mtDNA)
Approximately 16500 bp
Every bp make up part of coding sequence (no
introns)
Principally encode ptns or RNA molecules involved
in mitochondrial function (components of
mitochondrial respiratory chain)
Critical role in apoptotic cell death
Every cell contain 100s mitochondria 100s
mitochondrial Chr virtually all mitochondria are
inherited from mother (sperm head contain no or few
mitochondria)
GENETIC DISORDERS
Spectrum of inherited or congenital genetic disorders
classified as
Chromosomal disorders, including mitochondrial
chromosome disorders
The Mendelian disorders
Sex-linked single-gene disorders
Variety of non-Mendelian disorders & multifactorial
disorders all are result of mutation in genetic code
Chromosomal abnormalities are very common
1/2 spontaneous abortions have Chr abnormalities
Autosomal aneuploidy (differing from normal diploid
number) is severe > Sex Chr aneuploidies
ABNORMAL CHROMOSOME NUMBERS If Chr fail to separate (nondisjunction) either in
meiosis or mitosis 1 daughter cell will receive 2
copies of that Chr & 1 daughter cell will receive no
copies of that Chr
Non-disjunction can occur e autosomes or sex Chr
Chromosomal disorders
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If non-disjunction occurs during meiosis ovum or
sperm e either
Extra Chr trisomy (3 instead of 2 copies of Chr)
No Chr monosomy (1 instead of 2 copies of Chr)
Examples
Only trisomy 13, 18 & 21 (Downs syndrome)
survive to birth (most children e trisomy 13 &
18 die in early childhood)
Full autosomal monosomies extremely rare &
very deleterious
Sex Chr trisomies e.g. Klinefelters syndrome
(44+XXY) are relatively common
Sex Chr monosomy e.g. Turners syndrome
(44+X0)
Occasionally non-disjunction during mitosis shortly
after 2 gametes fused 2 cell lines each e different
Chr complement (more often e sex Chr) mosaicism
Very rarely entire chromosome set will be present
in >2 copies triploidy (69 Chr) or tetraploidy (92
Chr) spontaneous abortion
ABNORMAL CHROMOSOME STRUCTURES Abnormal Chr structures can disrupt DNA & genes
Deletions
Deletions of portion of Chr disease if 2 copies
of genes in deleted region are necessary (the
individual will not be normal e the 1 copy remaining)
Deletion Duplication
Inversion Balanced translocation
copy remaining on the non-deleted homologous)
Example
Prader Willi syndrome cytogenetic events
deletion of part long arm of Chr 15
Wilms tumour deletion of part of short arm
of Chr 11
DiGeorge syndrome microdeletions in long
arm of Chr 22
Duplications
When portion of Chr is present on the Chr in 2
copies genes in that Chr portion are present in
extra dose e.g. CharcotMarieTooth disease (form
of neuropathy) is due to small duplication of region
of Chr 17
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Inversion
End to end reversal of segment ein a chromosome
e.g. abcdefgh becomes abcfedgh (haemophilia)
Translocations
2 Chr regions join together (not normally do)
Chr translocations in somatic cells tumorigenesis
Translocations can be very complex involving >2 Chr
but most are simple & fall in 1 of 2 categories
Reciprocal translocation
o When any 2 non homologous Chr break
simultaneously & rejoin, swapping ends
o Cell still has 46 Chr (2 of them rearranged)
o Someone e balanced translocation is likely to
be normal unless the breakpoint interrupts a
o At meiosis when Chr separate in different
daughter cells translocated Chr will enter
gametes & any resulting fetus may inherit 1
abnormal Chr & have unbalanced
translocation e physical manifestations
Robertsonian translocation
o When 2 acrocentric Chr join & short arm is
lost only 45 Chr
o It is balanced translocation as no genetic
material is lost & the individual is healthy but
any offspring have risk of inheriting
unbalanced arrangement depending on w
acrocentric Chr is involved
o Clinically relevant is 14/21 Robertsonian
translocation in woman 1 in 8 risk of
having baby e Downs syndrome (male carrier
has 1 in 50 risk)
o 50% risk of producing carrier like
themselves genetic family study is
necessary
MITOCHONDRIAL CHROMOSOME DISORDERS
No introns in mitochondrial genes mutation has high
chance of having effect however as every cell contains
100s of mitochondria so single altered mitochondrial
genome is not noticed
As mitochondria divide likelihood of more mutated
mitochondria mitochondrial disease
Most mitochondrial diseases are myopathies &
neuropathies e maternal pattern of inheritance
Myopathies (CPEO) chronic progressive external
ophthalmoplegia
Encephalomyopathies (MERRF) myoclonic
epilepsy with ragged red fibres
MELAS mitochondrial encephalomyopathy, lactic
acidosis & stroke-like episodes
KearnsSayre syndrome ophthalmoplegia, heart
block, cerebellar ataxia, deafness & mental
deficiency due to long deletions & rearrangements
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28
(LHON) Lebers hereditary optic neuropathy
commonest cause of blindness in young men e
bilateral loss of central vision & cardiac
arrhythmias it is mitochondrial disease caused
by point mutation in one gene
Multisystem disorders Pearsons syndrome
(sideroblastic anaemia, pancytopenia, exocrine
pancreatic failure, subtotal villous atrophy, DM &
renal tubular dysfunction
Hearing loss may be the only symptom & 1 of
mitochondrial genes implicated predispose to
aminoglycoside ototoxicity
Other abnormalities retinal degeneration, DM &
hearing loss
ANALYSIS OF CHROMOSOME DISORDERS Cell cycle arrested at mitosis by colchicines staining
examine for abnormality
YAC-cloned probes labelled e fluorescently tagged
nucleotides in insitu hybridization
Mendelian & sex-linked single-gene disorders are due
to mutations in coding sequences & their control
elements
All cause dysfunction of the protein product
MUTATIONS Point mutation (Missense mutation)
The simplest type of change
Substitution of 1 nucleotide for another change
codon in coding sequence
Gene Defects
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29
Example triplet AAA (codes for lysine) mutated
to AGA (codes for arginine)
Whether it produces clinical disorder depends on
whether it change critical part of ptn molecule
produced
Many substitutions have no effect as several codons
code for same aa
Some mutations have severe effect e.g. in sickle cell
disease mutation in globin gene change 1 codon from
GAG to GTG valine is incorporated into polypeptide
chain (instead of glutamic acid) w radically alters its
properties
Insertion or deletion Insertion or deletion of 1 or more bases is more
serious as it alteration of rest of the following
sequence (frame-shift mutation)
Example
If the original code was
TAAGGAGAGTTT
Extra nucleotide (A) is inserted
TAAAGGAGAGTTT
If 3rd nucleotide (A) is deleted
TA-GGAGAGTTT
In both cases different aa incorporated in
polypeptide chain
It is responsible for some forms of thalassaemia
Missense mutation
Nonsense mutation
Insertions & deletions can involve 100s of bp of DNA
examples
Large deletions in dystrophin gene remove coding
sequences Duchenne muscular dystrophy
Insertion/deletion (ID) polymorphism in ACE gene
genotypes II, ID & DD deletion of 287 bp
repeat sequence & DD is associated e higher
concentrations of circulating ACE heart disease
Splicing mutations If DNA sequences w direct splicing of introns from
mRNA are mutated abnormal splicing
Processed mRNA w will be translated to ptns by
ribosomes may carry intron sequences altering w aa
are incorporated in polypeptide chain
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30
Termination mutations (Nonsense mutation) Normal polypeptide chain termination occurs when
ribosomes processing mRNA reach one of the chain
termination or stop codons
Mutations involving stop codons late or premature
termination
Example haemoglobin Constant Spring Hb variant
where instead of stop sequence single base change
insertion of extra aa
SINGLE-GENE DISEASE Monogenetic disorders involving single genes can be
inherited as dominant, recessive or sex-linked
Many syndromes show multiple forms of inheritance
pattern because multiple defects occur in given
disease associated gene or in separate genes for
example in EhlersDanlos syndrome AD, AR & XL
inheritance
Autosomal dominant disorders (AD) Overall incidence 7 in 1000 live births
AD disorder occurs when 1 of 2 copies of autosomal
Chr has mutation & ptn produced by normal gene
cannot compensate
Heterozygous individual e 2 different forms (or
alleles) of same gene manifest the disease
Offspring of heterozygotes 50% inheriting Chr
carrying disease allele also have the disease
Estimation of risk to offspring for counselling families
can be difficult because
Great variability in their manifestation
incomplete penetrance if patients have dominant
disorder but does not manifest clinically
appearance of the gene having skipped generation
Variable expression dominant traits are
extremely variable in severity e.g. mildly affected
parent may have severely affected child
New cases in previously unaffected family may be
due to new mutation risk of further affected
child is negligible e.g most cases of achondroplasia
are due to new mutations
Autosomal recessive disorders (AR) Overall incidence 2.5 in 1000 live births
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31
Manifest only when individual is homozygous for
disease allele i.e. both Chr carry the mutated gene
Parents are generally unaffected healthy carriers
(heterozygous for disease allele)
Usually no family history (although defective gene
pass from generation to generation)
Offspring of affected person is healthy carrier unless
the other parent is also carrier
If carriers marry offspring
1 in 4 chance homozygous & affected
1 in 2 chance (2 in 4) being a carrier
1 in 4 chance being genetically normal
Clinical features of AR disorders are usually severe,
patients present in 1st first few years of life & high
mortality
Sex-linked disorders o Genes carried on X-Chr said to be Xlinked &
can be dominant or recessive
o Females have 2 X-Chr unaffected carriers
of X linked recessive diseases
o Males have 1 X-Chr any deleterious
mutation in X linked gene will manifest (no
2nd copy of gene)
X linked dominant disorders (XLD)
Females e heterozygous mutant gene & males e 1
copy of mutant gene manifest the disease
Affected mother 1/2 male or female offspring
are affected
Affected father all female offspring are
affected & all male offspring are unaffected
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32
Affected males tend to have severe disease >
heterozygous female
X linked recessive disorders (XLR)
These disorders present in males & homozygous
female (usually rare)
Transmitted by healthy female carriers or
affected males if they survive to reproduce
Example of an XLR is haemophilia A (mutation in X
linked gene for factor VIII in 50% there is
intra Chr rearrangement (inversion) of tip of long
arm X-Chr one break point ein intron 22 of
factor VIII gene)
Offspring of carrier female + normal male
50% of girls are carriers inherit mutant allele
from their mother & normal allele from their
father
50% of girls inherit 2 normal alleles normal
50% of boys have haemophilia as they inherit
mutant allele from their mother (& Y Chr from
their father)
50% of boys are normal inherit normal allele
from mother & Y Chr from their father
Male e haemophilia + normal female normal male
offspring + carrier females
Y-linked genes
Genes carried on Y Chr are said to be Y linked
Only males are however no known examples of Y
linked single gene disorders
Sex-limited inheritance
Occasionally a gene can be carried on an autosome
but manifest only in one sex frontal baldness is
an AD in males but behave as AR in females
Other single-gene disorders These are disorders w may be due to mutations in
single genes but do not manifest as simple monogenic
disorders
They can arise from variety of mechanisms
Triplet repeat mutations
In gene responsible for dystrophia myotonica
mutated allele was found to have expanded
3UTR region in w three nucleotides (CTG) was
repeated up to 200 times
In families e dystrophia myotonica people e
late onset disease had 2040 copies of the
repeat but their children & grandchildren who
presented e disease from birth had increase
in number of repeats (up to 2000 copies)
number of triplets affects mRNA & ptn function
Mitochondrial disease
Imprinting
In some way (not yet clear), the fetus can
distinguish between Chr inherited from mother
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33
& Chr inherited from father (although both give
23 Chr)
The Chr are imprinted maternal & paternal
contributions are different
Imprinting is relevant to human genetic disease
because different phenotypes may result
depending on whether mutant Chr is maternal or
paternal
Deletion of part of long arm of Chr 15 (15q11
q13) PraderWilli syndrome if it is paternally
inherited but deletion of similar region of the
Chr Angelmans syndrome if it is maternally
inherited
The affected gene is identified as ubiquitin
(UBE3A)
Significantly maternal Chr 15 UBE3A is
expressed in brain & hypothalamus defective
maternal ubiquitin in Angelmans syndrome
accumulation of undegraded ptn & neuronal
damage
COMPLEX TRAITS: MULTIFACTORIAL & POLYGENIC INHERITANCE
Combination of genetic & environmental factors are
said to be multifactorial
Those involving multiple genes are said to be polygenic
Measurements of most biological traits e.g. height is
variant thought to be due to additive effects of
number of alleles at number of loci many of w are
individually identified using molecular biological
techniques
There are sex differences e.g. congenital pyloric
stenosis is most common in boys but if it occurs in
girls larger number of affected relatives
Most human diseases e.g. heart disease, DM and
common mental disorders are multifactorial traits
Aims of genetic counselling
Obtain full history pregnancy history, drug,
alcohol ingestion during pregnancy & maternal
illnesses
Establishing accurate diagnosis of genetically
abnormal child
Genetic Counselling
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34
Draw family tree & questions about abortions,
stillbirths, deaths, marriages, consanguinity
Estimate risk of future pregnancy being affected
Give information about prognosis & management
Continued support & follow-up
Genetic screening including prenatal diagnosis
PRENATAL DIAGNOSIS Should be offered to all pregnant women in UK but it
is offered to high risk mothers only
Investigations depend on gestation 711 Weeks
Vaginal U/S
Confirm viability, fetal number & gestation by
crown rump measurement
1113 Weeks & 6 days (combined test)
U/S for nuchal translucency measurement (normal
fold triple test
alone at 16 weeks
All serum marker are corrected for gestational
ages multiple of the mean (MOM) value for
the appropriate gestation week is necessary
Chorionic villus sampling (CVS) at 1113 weeks under
U/S control to sample placental site
Amniocentesis at 15 weeks to sample amniotic fluid
1420 Weeks (serum triple or quadruple test)
The triple test for Chr abnormalities testing
maternal serum for
-fetoprotein (low) in neural tube defects
Unconjugated oestradiol (low)
Human chorionic gonadotrophin (high) for
Downs syndrome & neural tube defects
The quadruple test
The triple test + inhibin-A ( in Downs
syndrome)
If too late for triple test or previous option not
offered
1422 Weeks
U/S for structural abnormalities e.g. neural tube
defects, gestation period
The best time to detect congenital heart defects
is 1822 weeks
Reported detection rates for all congenital defects
vary from 14 to 61% for hypoplastic ventricle to
97-100% for anencephaly
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35
Gene therapy entails placing normal copy of gene into
the cells of patient who has defective copy of the
gene (concentrating on recessive disorders e.g. cystic
fibrosis where the disease is due to absence of normal
gene product)
In dominant disorders it is difficult & complicated
2 major factors are involved in gene therapy
Introduction of functional gene sequence in target
cells
Expression & permanent integration of transfected
gene in host cell genome
Suitable diseases for current gene therapy include
Cystic fibrosis
CFTR gene
o Cystic fibrosis transmembrane regulator
gene is the responsible for cystic fibrosis
o It was 1st localized to Chr 7 by linkage
analysis
o CFTR gene spans about 250 kbp & contains
27 exons
o DNA sequence analysis predicts polypeptide
sequence of 1480 aa
o CFTR gene also encodes a simple Cl- ion
channel
Mutation
o The commonest is single mutation e 3 bp
deletion in exon 10 removal of codon
specifying phenylalanine (F508del)
o Also >1000 different minor mutations of
CFTR gene e most mapping to ATP-binding
domains
Gene therapy experiments
o Still under trial to restore CFTR function by
transfection of cells e wild type receptor
o 2 different routes are tried
Placing CFTR gene in adenovirus vector
Placing CFTR gene in liposome (conveyed
to lung by aerosol spray) fatty surface
of liposome fuses e cell membrane to
deliver CFTR DNA into cell
o Topical nasal gentamicin (aminoglycoside AB)
expression of functional CFTR channels
Adenosine deaminase (ADA) deficiency
Rare immunodeficiency disease introducing
normal human ADA gene in patients
lymphocytes reconstitute function of cellular
& humoral immunity in severe combined
immunodeficiency
Familial hypercholesterolaemia
It is due toLDL receptor gene
Gene Therapy
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36
Gene therapy receptor gene is inserted in
hepatocytes (removed by liver biopsy) gene-
corrected hepatocytes reinjected in portal
circulation migrate back to liver
reincorporated start to produce LDL
receptor protein dramatically cholesterol
level
TREATMENT OF SOMATIC DISEASE Vascular disease
Neovascularization toblood flow & repair cardiac
tissue after MI temporary expression of angiogenic
factors at site of blockage new blood vessels
Local temporary expression of clot disintegrating
enzymes e.g streptokinase & lipases repair damaged
& diseased arteries
Deliver liposomes loaded e DNA or direct inject of
DNA plasmids to tissue ptn will be expressed by
cells (only 13% but it is sufficient for local effect
required)
Neuronal disease Neurotrophic factors can be transiently expressed
same as e vascular diseases nerve cell regeneration
& maintenance
Extend expression period of neurotrophin by injecting
transfected myocytes in damaged area fuse e any
adjacent muscle
Cancer Cancer is genetic disease & many genes are
deregulated
p53 is TSG apoptosis in cells e damaged genetic
material reintroduction & overexpression of
functional p53 in tumours is investigated
Since it is only likely to occur in rapidly dividing cells
perfect target for cancer gene therapy by repeat
exposure to vectors e.g. retroviruses, liposomes &
naked DNA plasmids
Tumour growth depends on development of new blood
vessels (angiogenesis) & inhibitors are under trial
Stem cell therapy Number of adult stem cell therapies already exist
particularly bone marrow transplants
It is anticipated to treat wide variety of diseases
require replacement of destroyed tissues e.g.
Parkinsons, spinal cord injuries & muscle damage
The blood in umbilical cord is available & rich source of
haemopoietic stem cells i.e. CD34 +ve & CD38 ve
colonize bone marrow & rapidly populating marrow e all
various cells (RBCs & WBCs)
Umbilical cord stem cell, dubbed cord blood-derived
embryonic like stem cells (CBEs) able to
differentiate to more types of tissue not simply
haemopoietic cells (super pluripotentiality)
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37
Primitive monocyte derived multipotential cell (MOMC)
could be isolated from adult peripheral circulating
monocytes induced (given the correct paracrine,
environmental & adhesion signals) endothelia,
neurones, cardiomyocytes & mesenchymal lineages
Similar reports concerning adult stem cells isolated
from skin
THE HUMAN PROTEOME PROJECT Studying of ptn expression characteristics of normal
& diseased cells
Achieved by using 2D gel electrophoresis
Pattern of dots corresponds to different ptn
expressed non-, over- & underexpression of given
ptn can be detected by corresponding change on
proteome
Post-translational modifications of ptns show up as
change in either size or charge on proteome picture
2D gel electrophoresis comparing paired serum & synovial fluid in patient e RA. The circled ptns indicate major ptns w differ between the 2 biofluids.
Although serum contained many ptns not found in synovial fluid & 1 major ptn was found in synovial fluid but not in serum. This indicates that synovial fluid
is not simple transudate (exudate)
Cancers are genetic diseases & involve changes to
normal function of cellular genes
Multiple genes interact during oncogenesis & stepwise
progression of defects leads over proliferative of
particular cell to full breakdown of control ( apoptosis)
Susceptibility to development of particular form of
cancer can be inherited
Cancer tissues are clonal & arise from changes in only
one cell w then proliferates in the body
The genes that are primarily damaged by genetic
changes w lead to cancer fall in 2 categories:
oncogenes & TSG
Oncogenesis is multistep process number of
mutations or alterations to key genes are required
before malignant phenotype is expressed
Once mutations begun to cause unchecked clonal
expansion of 1ry tumour cells further mutations
occur ein subsequent generations of daughter cells
clones w are invasive & or form metastases
ONCOGENES Genes coding for growth factors, growth factor
receptors, 2ry messengers or even DBP would act as
promoters of abnormal cell growth if mutated
The Genetic basis of Cancer
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Viruses carry genes w when integrated to host cell
promote oncogenesis (v-oncogenes) & later their
normal cellular counterparts (c-oncogenes) were found
Oncogenes encode ptns that participate in regulation
of normal cellular proliferation e.g. erb-A on
chromosome 17q11q12 encodes for thyroid hormone
receptor
Examples of acquired/somatic mutations & proto-oncogenes
Point mutation K-ras
DNA amplification Myc HER2-neu
Chromosome translocation BCR-ABL PML-RAR Bcl-2/IgH c-myc & Ig
Pancreatic cancer
Neuroblastoma Breast cancer
CML, ALL APML Follicular lymphoma Burkitts lymphoma
CML, chronic myeloid leukaemia; ALL, acute lymphoblastic leukaemia; APML, acute promyelocytic leukaemia
Activation of oncogenes Non activated oncogenes w are functioning normally
(proto-oncogenes)
Transformation to oncogenes can occur by 3 routes
Mutation
Carcinogens e.g. cigarette smoke, ionizing
radiation UVR can cause point mutation in
genomic DNA
By chance some of these point mutations will
occur in regions of oncogene activation of
that gene
Not all bases in oncogene cause cancer if
Mutated but some do (those in coding region)
Chromosomal translocation If during cell division an error occurs & 2 Chr
translocate portion swaps over
translocation breakpoint in middle of 2 genes
If this happens end of 1 gene is translocated
on to beginning of another gene (fusion gene)
sequences of 1 part of fusion gene are
inappropriately
Example of fusion gene (Philadelphia Chr) in
GML
Similarly in Burkitts lymphoma translocation
replace the regulatory segment of myc
oncogene by regulatory segment of unrelated Ig
Viral stimulation When viral RNA is transcribed by RT to viral
cDNA & in turn spliced in cellular DNA viral
DNA may integrate & activate oncogene
Alternatively the virus may pick up cellular
oncogene DNA & incorporate it to its own viral
genome
Subsequent infection of another host cell may
expression of this viral oncogene e.g. Rous
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sarcoma virus of chickens was found to induce
cancer because it carried ras oncogene
After the initial activation other changes occur
in DNA
TUMOUR SUPPRESSOR GENES (TSG) These genes restrict undue cell proliferation (in
contrast to oncogenes) & induce repair or self
destruction (apoptosis) of cells contain damaged DNA
Example germline mutations in genes found in non-
polyposis CRC responsible for repairing DNA
mismatches
1st TSG to be described was RB gene mutations in
RB Retinoblastoma
1 in 20000 young children
Familial variety of retinoblastoma 1st mutation is
inherited & by chance 2nd somatic mutation occurs
e the formation of tumour
Sporadic variety of retinoblastoma by chance
both mutations occur in both RB genes in a single
cell
Other TSG gene p53
Mutations in p53 have been found in almost all
human tumours including sporadic CRC, carcinomas
of breast & lung, brain tumours, osteosarcomas &
leukaemias
The ptn encoded by p53 is cellular 53 kDa nuclear
phosphoprotein (plays role in DNA repair &
synthesis in control of cell cycle, differentiation &
apoptosis)
p53 is DBP
Activate many gene expression pathways but it
is normally only short lived
p53 is likely to act as tetramer mutation in
single copy of gene can promote tumour
formation because hetero tetramer of mutated
& normal p53 subunits would still be
dysfunctional
In many tumours mutations that disable p53
function also prevent its cellular catabolism
although in some cancers there is loss of p53 from
both Chr in most cancers (particularly CRC) such
long lived mutant p53 alleles can disrupt normal
alleles ptn
How TSG work?
TSG products are involved in control of cell cycle
Progression through cell cycle is controlled by many
molecular gateways w are opened or blocked by cyclin
group of ptns that are specifically expressed at
various stages of the cycle
RB & p53 proteins control cell cycle & interact
specifically e many cyclin ptns (The latter are
affected by INK 4 acting on p16 ptns)
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General principle being held at 1 of these gateways
programmed cell death
p53 induces expression of other genes & its own
expression is induced by broken DNA initially cause
expression of DNA repair enzymes, if repair is too
slow or cannot be effected then other ptns induced by
p53 will effect programmed cell death
Viral inactivation of tumour suppressors Suppression of normal TSG function by disabling
normal ptn (once it is transcribed) rather than by
mutating the gene
Viruses have developed their own genes w produce
ptns to do precisely this
The main targets of these ptns are RB & p53 to w
they bind & disable
Adenovirus E1A & HPV E7 gene products bind RB
Adenovirus E1B & HPV E6 gene products bind p53
SV40 virus large T Ag binds both RB & p53
Microsatellite instability Microsatellites are short (50300 bp) sequences
composed of tandemly repeated segments of DNA
2-5 nucleotides in length (di/tri/tetranucleotide
repeats) scattered throughout the genome in non-
coding regions between genes or ein genes (introns)
Many of these microsatellites are highly
polymorphic
Often used as markers for linkage analysis because
of high variability in repeat number between
individuals
These regions are inherently unstable & susceptible
to mutations
Somatic microsatellite instability (MSI) has been
detected in number of tumours
Detecting MSI involve comparing length of
microsatellite alleles amplified from tumour DNA e
the corresponding allele in normal tissue from same
individual
Recent studies indicate that MSI can be detected
in 90% of tumours from individuals e hereditary
non-polyposis CRC
The presence of these additional microsatellite
alleles (repeated segments) in tumour cells results
from inherent susceptibility of these areas to such
alterations & from mutations in DNA mismatch
repair mechanism that would normally correct
these errors
Tumour angiogenesis Once a nest of cancer cells reaches 12 mm in
diameter it must develop blood supply in order to
survive & grow as diffusion is no longer adequate to
supply the cells e O2 & nutrients
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As e all tissues, solid tumour cancer cells secrete
substances that promote formation of new blood
vessels (angiogenesis)
Substances identified to promote angiogenesis e.g.
angiopoietin-1, basic fibroblast growth factor
(bFGF) & vascular endothelial growth factor (VEGF)
Inhibitors of angiogenesis (part of cancer
treatment strategy)
Angiostatin polypeptide of 200 aa produced
by cleavage of plasminogen & binds to subunits
of ATP synthase exposed at surface of cell
embedded in plasma membrane
Endostatin polypeptide of 184 aa w is derived
from globular domain found at the C-terminal of
type XVIII collagen (specific collagen of blood
vessels) cleaved from the parent molecule
Several therapeutic vaccine preparations are under
development to produce range of host immunity
responses (humoral & cellular) against pro-
angiogenic factors & their receptors in tumours
1 approach has been directed at cell adhesion
molecules found in tumour blood vessels
Vitaxin monoclonal Ab against alpha-v/beta-3
vascular integrin shrinks tumours in mice eout
harming them