Basic Genetics for MRCP part 1 all you are looking for

1. ٢٠١٣/١٠/١٩ ‫ﺍﻟﺳﺑﺕ‬
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 Cell 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 H2Osoluble molecules
Non-polar
(insoluble)
lipid
hydrophobic
tail
(commonly 2 long-chain FA)
1

2. •
CELL DYNAMICS
•
Old cellular ptn mopped up by small cofactor molecule
It is implicated in large number of human diseases
•
When free radical reacts e’ non-radical
reaction
(ubiquitin)
chain
direct tissue damage by membrane lipid
peroxidation
Small 8.5 kDa regulating ptn
•
Present universally in all living cells
The major free radical species produced in human
Interacts e’ these worn ptn via their exposed
body
hydrophobic residues
1) Hydroxyl radical (OH)
A complex containing >5 ubiquitin molecules is
The most reactive but others can generate
rapidly degraded by large proteolytic multienzyme
more reactive species as breakdown products
‘26S proteosome’
Can
Plays role in regulation of receptor tyrosine kinase
purines & pyrimidines
Failure to remove worn proteins
genetic
mutations
by
attacking
(SOD)
convert
2) Superoxide radical (O2-)
in cell cycle & repair of DNA damage
•
cause
Superoxide
chronic debilitating
dismutases
disorders e.g. Alzheimer & frontotemporal dementias
(accumulation of ubiquinated ptn w’ are resistant to
antioxidant mechanism)
ubiquitin-mediated proteolysis)
•
superoxide to hydrogen peroxide (protective
Pt e’ dominant familial forms of amyotrophic
Resistant ubiquinated proteins
found in myositis & myopathies
lateral sclerosis (MND)
inclusion bodies
mutations in gene for
Cu–Zn SOD-1 catalases
causes
Point mutation in target ptn itself e.g. mutant p53
Glutathione peroxidases
in cancer
hydrogen peroxide & generated by SOD in cell
External factor altering normal ptn conformation
cytosol & mitochondria
3) Nitric oxide (NO)
proteolytic-resistant shape e.g. CJD
•
✰ Free radicals
•
It is any atom or molecule w’ contains 1 or more
unpaired electrons
species
more reactive than the native
enzymes remove
Alpha-tocopherol,
urate,
ascorbate
&
glutathione
remove free radicals by reacting directly & noncatalytically
↓ α-tocopherol ( ↓ vitamin E)
neurodegeneration
2

3. •
The principal dietary antioxidants are vitamin E,
vitamin C, β-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
•
✰ Pinocytosis
HSPs are expressed in a wide range of human cancers
&
implicated
in
tumour
cell
proliferation,
differentiation, invasion, metastasis, cell death &
immune response
•
Much smaller-scale model of phagocytosis
•
Continually occurring in all cells
•
In contrast to phagocytosis
molecular complexes e.g. LDL
surface clumping &
internal accumulation of a protein called clathrin
PHAGOCYTOSIS, PINOCYTOSIS
& EXOCYTOSIS
•
✰ Phagocytosis
•
Clathrin-coated pits pinch inwards as clathrin-coated
vesicles
•
Specialized cells e.g. macrophages & neutrophils
•
Lysosomes rapidly fuse e’ phagosomes
equally rapid
digestion of contents & recycling
•
receptors for smaller
Clathrin prevents fusion of lysosomes (removal
lysosomal fusion & degradation)
✰ Exocytosis
•
Maintenance of clathrin coat
transcellular transit
Only triggered when specific cell surface receptors
of contents & their exocytosis at another side of
(macrophage Fc receptor)
plasma membrane i.e. apical to basal transcytosis
occupied by their ligand
3

4. •
Some of these vesicles rapidly fuse e’ plasma
2) Carrier proteins
membrane & exocytose their contents
•
Other vesicles do not immediately fuse e’ plasma
Shuttling the solute across
membrane
•
Slower in action
Facilitating diffusion down a gradient across the
The clathrin-coated vesicles
have additional lipid
membrane OR actively pumping solutes against
bilayer embedded proteins called v-SNAREs (signal &
response elements)
the gradient using ATP as energy
interact e’ target organelle
membrane proteins called t-SNAREs
vesicle fusion
is therefore specific in the correct place & in the
RECEPTORS
•
correct time e.g. neuronal transmitter vesicles
Membrane surface receptors pass their EC signal
across plasma membrane to cytoplasmic 2ry signalling
molecules
•
MEMBRANE TRANSPORT & ION CHANNELS
•
Membrane-bound receptors is subclassified according
to mechanism by which they activate signalling
Plasma membrane is freely permeable to
molecules
Gases e.g. O2, CO2 and N2
+
−
Small uncharged molecules e.g. H2O (not H & OH )
Ion channel linked
& urea
G-protein linked
Larger hydrophobic lipid-soluble molecules e.g.
Enzyme linked
steroids
•
•
Structure of plasma membrane receptors
Serpentine
small charged ions (K, Na, Ca, Cl, Mg & HCO3) cannot
receptor
pass unless via specific transport ptn embedded in
Transmembrane with large EC & IC domains e.g.
plasma membrane
•
Large uncharged molecules (G, aa & nucleotides) and
7 transmembrane domains e.g. LH
EGF receptor
2 Structural types of transport molecules/complex
Transmembrane with large EC domain only e.g.
1) Channel proteins
macrophage scavenging receptors
Open a channel in the lipid membrane
Entirely linked to outer membrane leaflet by lipid
Allow specific solute to pass through
moiety known as GPI anchor (glycan phosphatidyl
inositol) e.g. T cell receptor
4

5. •
Function of membrane receptors is to initiate 2ry
Specifically phosphorylate kinases on small set
message
of IC signalling proteins OR associate e’ ptn e’
activation of specific enzyme or DNA-
tyrosine kinase activity
binding protein. This may involve
✰ G-protein-linked receptors
•
Once activated by ligand
β, γ)
•
3) Tyrosine phosphatase receptors
e.g. CD45
binds trimeric complex (α,
Remove phosphates from tyrosine residues of
anchored to inner surface of plasma membrane
specific IC signalling proteins
The complex is GTP-binding protein or G-protein then
4) Serine/threonine kinase receptors
interacts e’ enzyme complexes anchored to inner
leaflet of the membrane
•
e.g. TGF-β receptor
These complexes
Phosphorylate
1 or all 3 of 2ry messengers
cyclic AMP (cAMP)
Ca2+ ions
Inositol trisphosphate / diacylglycerol (IP3/DAG)
serine
&
threonine
residues of IC signalling proteins
•
Many IC receptors that bind lipid-soluble ligands e.g.
steroid hormones (Pg, cortisol), T3/T4
✰ Enzyme-linked surface receptors
•
specific
shape in response to binding their ligands
These receptors usually have single transmembrane
often change
enter the
nucleus & interact directly e’ specific DNA sequences
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 (Gkinase)
binds to & phosphorylates serine &
threonine residues of specific 2ry messengers
2) Tyrosine kinase receptors
Cytoplasm
•
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
e.g. PDGF receptor
5

6. •
It may contain ribosomes on the surface (rough
•
Lysosomal action is crucial to function of macrophages
endoplasmic reticulum ‘RER’) & when absent (smooth
endoplasmic reticulum ‘SER’)
•
& PMNs in killing & digesting infective agents, tissue
remodelling
ER is involved in processing of ptn
chain
•
development
&
osteoclast
remodelling of bone
ribosomes
translate mRNA to 1ry sequence of aa of ptn peptide
during
✰ Peroxisomes
•
This chain is synthesized in the ER where it is folded
Dense cellular vesicles
contain enzymes catalyse the
breakdown of H2O2
& modified into mature peptides
•
•
They are involved in metabolism of bile & FA
ER is the major site of drug metabolism
•
Primarily concerned e’ detoxification e.g. d-amino acid
✰ Golgi apparatus
oxidase & H2O2 catalase
•
•
Consist of flattened cisternae similar to ER
•
Characterized as stack of cisternae from w’ vesicles
rare metabolic disorders
e.g. Zellweger’s syndrome & rhizomelic dwarfism
✰ Mitochondria
bud off from the thickened ends
•
The inability to function
The 1ry processed peptides of ER are exported to
•
The powerhouse of the cell
Golgi apparatus for maturation into functional ptn e.g.
•
Each mitochondrion has 2 lipid bilayer membranes
glycosylation of ptn to be excreted before packaging
•
The outer membrane
into secretory granules & cellular vesicles that bud off
It contain many gated receptors
the ends
materials like pyruvate & ADP
✰ Lysosomes
•
Dense
Fuse
cellular
e’
membrane
vesicles
contain
acidic
digestive
Proteins of Bcl-2/Bax family are incorporated in
the outer membrane
phagocytotic
vesicles
from
outer
cell
digest contents into small biomolecules
capable of cross lysosomal lipid bilayer to cytoplasm
•
oxaloacetate &
ATP
enzymes
•
import raw
Lysosomal enzymes can be released outside cell by
can release mitochondrial
enzymes that trigger apoptosis
•
The inner membrane
Highly infolded to form cristae to↑its effective
surface area
fusion of the lysosome e’ plasma membrane
6

7. Cofactors in cAMP response element binding
Contains transmembrane enzyme complexes of
electron transport chain
gradient
generate H
+
ion
phosphorylation pathway
drives adjacent transmembrane ATPase
•
complex to form ATP from ADP & Pi
•
proteins (CREB) are co-activated & interact e’
The inner matrix
Other
G-protein
complexes
activate
membrane bound phospholipase complexes
inner
cleave
It possesses several copies of its own DNA in
membrane phospholipid-polyphosphoinositide (PIP2)
circular genome
1) Inositol
trisphosphate
H2O
(IP3)
It contains enzymes of Krebs cycle that generate
molecule
substrates
ion channels in ER (or sarcoplasmic reticulum in
of
both
electron
transport
chain
(FADH2 & NADH) & central metabolism e.g.
floats in cytoplasm
soluble
muscle cells)
succinyl CoA, α-oxoglutarate, oxaloacetate
rapid release of Ca2+
2) Diacylglycerol (DAG)
membrane
interacts e’ gated
lipid soluble that remains at
activates a serine/threonine kinase
protein kinase C
Secondary Messenger
•
The cellular calcium-binding proteins & ion pumps
rapidly remove Ca2+ from cytoplasm back into storage
•
2ry messengers are molecules that transduce a signal
from a bound receptor to its site of action
•
compartment e.g. ER
•
Free Ca2+ interacts e’ target proteins in cytoplasm
There are essentially 4 mechanisms by which 2ry
phosphorylation
messengers act (cross talk & rarely activated alone)
activated DNAbinding proteins entering nucleus
cAMP
Ca2+ ions
IP3/DAG
Protein phosphorylation
✰ cAMP, IP3/DAG & Ca2+ ions
•
Generation of cAMP by G-protein-linked receptors
↑ cellular cAMP
binding proteins
bind & activate specific cAMP
dimerize & enter nucleus
/
dephosphorylation
cascade
✰ 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
interact e’ set DNA sequences (cAMP response
elements)
7

8. •
NFκB
conformational change & enter nucleus
initiates transcription of specific genes
•
Lipid-soluble ligands e.g. steroids
messengers
not need 2ry
cytoplasmic receptors once activated
enter nucleus as DBP
alter gene expression directly
The Cytoskeleton
•
Complex network of structural ptns w’ regulates
Shape of the cell
Cell ability to traffic internal cell organelles &
move in response to external stimuli
•
Inner membrane & cytoplasmic targets of these
activated receptor complexes are ras, ptn kinase C &
•
The major components
1) Microtubules
ultimately MAP (mitogen activated ptn) kinase, Janus-
Made of 2 ptn subunits
Stat pathways or phosphorylation of IκB
Continuously
release its
change
α & β tubulin (50 kDa)
length
‘highway’
DNA-binding protein, nuclear factor kappa B (NFκB)
IC signalling proteins usually contain conserved non-
2 motor microtubule associated ptns (dynein &
catalytic regions called SH2 & SH3 (SRC homology
kinesin)
regions 2 & 3)
•
transporting organelles through cytoplasm
(dynein also
SH2 region binds to phosphorylated
antegrade & retrograde movement
beating of cilia)
tyrosine & SH3 domain is implicated in recruitment of
intermediates that activate ras proteins
•
During interphase
by microtubule organizing centre (MTOC) w’
Like G-proteins
ras (& its homologous family
consists of centrosomes containing tubulin &
switch between inactive GDP-
provide structure on w’ daughter Chr can
members rho / rac)
binding state & active GTP-binding state
microtubules rearranged
separate
8

9. 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
These filaments also present in nonmuscle cells
They make cell-to-cell contacts e’ adjacent cells
as truncated myosins (e.g. myosin 1), in cytosol
via desmosomes
(forming contractile actomyosin gel) & beneath
They make contact e’ basement matrix via
plasma membrane
hemidesmosomes
Cell movement is mediated by anchorage of
Function
actin filaments to plasma membrane at adherent
structural integrity (prominent in
cellular tissues under stress)
junctions
between
cells
non
stressed
Intermediate filament fibre ptns are specific to
coordination of contraction between adjacent
embryonic lineage of the cell e.g. keratin
cells of tissue (similarly, vertical contraction of
intermediate fibres only found in epithelial cells
tissues is anchored across cell membrane to
basement matrix at focal adhesion junctions
3) Microfilaments
Muscle cells contain
where actin fibres converge)
o Actin
Actinbinding ptns e.g. fimbria
highly ordered structure of actin
(globular ptn, 42–44 kDa)
o Myosin filaments
form contractile system
modulate
behaviour of microfilaments & their effects are
often Ca dependent
9

10. Actin-associated
specific e.g.
ptns
can
be
tissue
type
actin-binding troponin is complex
of 3 subunits & 2 of these have isomers w’ are
only found in cardiac muscle
•
reabsorbed)
abnormal Mg reabsorption of
Gitelman’s syndrome
2) Adherent junctions (zonula adherens)
Continuous on basal side of cells
Alterations in cell’s actin architecture are controlled
Contain cadherins
by activation of small ras-like GTP-binding proteins
The
rho & rac
microfilaments
division
involved in rearrangement of cell during
dysfunctions of these ptns are associated e’
malignancy
major
site
of
attachment
of
IC
Intermediate filaments attach to desmosomes
areas of thickened membranes of 2 adjacent
cells
Hemidesmosomes attach cells to basal lamina &
Intercellular Connections
also connected to intermediate filaments
Transmembrane integrins link EC matrix to
•
microfilaments at focal areas where cells also
tissues
•
EC domains form junctions between cells to form
attach to their basal laminae
Types of junction between cells
In blistering skin disorders auto-Ab
1) Tight junctions (zonula occludens)
by attacking tight junction desmosomal proteins
Situated at ends of margins adjacent to
e.g.
epithelial cells e.g. intestinal & renal cells
damage
desmoglein-1 in pemphigus foliaceus
Form barrier to movement of ions & solutes
desmoglein-3
in
pemphigus
vulgaris
&
3) Gap junctions
across the epithelium (may be variably leaky to
Allow substances to pass directly between cells
certain solutes)
e’out entering ECF
The ptns responsible for intercellular tight
Ptn channels (connexins) are lined up between 2
junction
adjacent cells & allow solutes passage up to MW
closure
(claudins)
selective
expression e’in tissue & regulate w’ ions pass
1000 kDa e.g. aa, sugars, ions, messengers
Mutations of claudin-16 (expressed in thick
Channels diameter is regulated by IC Ca2+, pH &
ascending
voltage
loop
of
Henle
where
Mg
is
10

11. Connexins
6 subunits surrounding channel &
The aa sequence arginine–glycine–aspartic acid
their isoforms in tissues are encoded by
(RGD)
different genes
integrin binding
Mutant connexins
disorders e.g. X-linked
form of Charcot–Marie–Tooth disease
potent recognition sequence for
Integrins replace cadherins in focal membrane
anchorage of hemidesmosomes & focal adhesion
junctions
Cell Adhesion & Molecules
The active form of integrin can come as result
of cytoplasmic signal that causes conformational
•
Major families of cell adhesion molecules
change in EC domain
↑affinity for its ligand
1) Cadherins
o The
signalling
‘inside-out’
occurs
when
Cadherins establish molecular links between
leucocytes
adjacent cells
peptides
They form zipper-like structures at ‘adherens
Ig super families structures e.g. Fc portion
junctions’
of Ig immunoglobulin
Through these junctions, bundles of actin
are
stimulated
by
bacterial
↑leucocyte integrin affinity for
o The ‘outside-in’ signalling follows binding of
filaments run from cell to cell.
ligand to integrin & stimulate 2ry signals
Related molecules e.g. desmogleins form the
diverse events e.g endocytosis, proliferation
main constituents of desmosomes (anchoring
& apoptosis
sites for intermediate filaments)
Defective integrins are associated e’ many
The expression of specific adhesion molecules in
immunological & clotting disorders e.g. Bernard–
the embryo is crucial for cell migration &
Soulier syndrome & Glanzmann’s thrombasthenia
differentiation of tissues
3) Ig superfamily cell adhesion molecules (CAMs)
2) Integrins
They are membrane glycoproteins e’
Ig-like structures domains
α & β
Neural cell adhesion molecule (N-CAM)
subunits w’ exist in active & inactive forms
o Predominantly in nervous system
They principally bind to EC matrix components
o Mediates homophilic adhesion
e.g. fibrinogen, elastase & laminin
11

12. o When bound to identical molecule on another
cell
N-CAM associate laterally with
fibroblast
growth
factor
The Nucleus & its responses
receptor
stimulate tyrosine kinase activity of that
•
A nucleus is present in all eukaryotic cells that divide
receptor
•
Contains human genome & bound by 2 bilayer lipid
growth of neurites (Adhesion
molecules can trigger cellular responses by
indirect
activation
of
other
types
of
membranes, the outer is continuous e’ ER
•
Nuclear pores present in membranes
allow passage
receptors)
o
of nucleotides & DNA interacting ptns in AND mRNA
The placenta and gastrointestinal
out
Placenta & GIT also express Ig superfamily
members but unclear function
•
The genome consists of DNA plus all apparatus for
replication & transcription into RNA
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 α granules of
platelets & Weibel–Palade bodies of endothelial
cells
it
moves
plasma
membrane
upon
stimulation of these cells
All 3 selectins play part in leucocyte rolling
12

13. •
•
Types of cell division
Meiosis
After stimulation from pro-mitotic EC signal e.g.
growth factor
G1 cyclin–Cdk complexes (CycB
Occurs in germ cells only
/Cdk4/6; CycE/Cdk2) become active to prepare cell
Chromosome complement is halved (haploid) & at
for S phase
fertilization the union of 2 cells restores full
expression of S cyclins (CycB/Cdk2) & enzymes
complement of 46 chromosomes
required for DNA replication
•
Mitosis
expression of transcription factors
G1 cyclin–Cdk complexes
degradation of molecules
Occurs in dividing cells after fertilization
that function as S phase inhibitors by targeting them
Results in 2 identical daughter cells
for ubiquitination
•
Chromosomes are only visible during cell division
•
A nucleolus is dense area e’in the nucleus
ptns & RNA
•
rich in
Active S cyclin–Cdk complexes phosphorylate ptns
that make up pre-replication complexes assembled
synthesis of rRNA & ribosomes
during G1 phase on DNA replication origins
serves 2
purposes
THE CELL CYCLE
•
1) Activate each already assembled pre-replication
Cells in quiescent G0 phase (G, gap) of the cycle are
complex
stimulated by receptor-mediated actions of growth
factors e.g. EGF, PDGF, IGF via IC 2
•
Stimuli
are
transmitted
transcription factors
to
nd
messengers
nucleus
2) Prevent new complexes from forming
•
activate
initiation of DNA synthesis
This ensures that every portion of genome will be
replicated once only
•
Mitotic
cyclin–Cdk
complexes
e.g.
CycB/CdK2
then mitosis & cell division
•
(synthesized but inactivated during G2 phase)
Cell cycling is modified by cyclin family of ptns
initiation of mitosis by stimulating downstream ptns
✰ Cyclin & cyclin-dependent kinases
•
assembly
Coordinated cyclic expression of cyclin-dependent
kinases (Cdk) drives cell replication cycle
•
involved in chromosome condensation & mitotic spindle
Cell cycle is catalysed by Cdk w’ are activated by class
of ptns called cyclins (Cyc)
✰ Apoptosis (programmed cell death)
•
Deliberate activation of constituent genes responsible
for their own demise
13

14. •
Necrotic cell death
Wound healing
External factor e.g. hypoxia, toxins damages cell’s
Normal metabolic processes e.g. autodestruction of
physiology
endometrium to cause menstruation
cell disintegration
Influx of water & ions
cellular organelles swell
Chemotherapy & radiotherapy only work if they can
rupture
Cell lysis
trigger tumour cells own apoptotic pathways
release of lysosomal enzymes in EC
environment
acute inflammatory responses in
vivo
•
•
Several factors initiate apoptosis but in general there
are 2 signalling pathways
1) The extrinsic pathway
Apoptotic cell death
Involved in processes e.g. tissue remodelling &
Chromatin aggregation + nuclear & cytoplasmic
induction of immune selftolerance
condensation in distinct membrane bound vesicles
Triggered by death receptors on cell surface e’
(apoptotic bodies)
internal death domain complexes
Organelles remain intact
caspase 8 molecules
Cell ‘blebs’
8
intact membrane vesicles
multiply pro-
release of initiator caspase
cleaves pro-caspase 3
caspase 3 + other
No inflammatory response
Cellular ‘blebs’ & remains are phagocytosed by
condensation & fragmentation
adjacent cells & macrophages
•
effector caspases
Death receptors are members of TNF receptor
2+
This process requires energy (ATP) and several Ca
Mg2+ dependent nuclease systems activation
&
cleave
superfamily
include CD95 (APO-1/Fas), TRAIL
(TNF-related
apoptosis
ligand)-R1,
TRAIL-R2,
TNF-R1, DR3 & DR6
nuclear DNA at the inter-histone residues
•
activate DNA cleavage, cell
Endonuclease destroys DNA following apoptosis
this
2) The intrinsic pathway
involve enzyme caspase (cysteine-containing aspartase-
Initiated at the mitochondrial level
specific protease) w’ activate CAD (caspaseactivated
release of cytochrome C from mitochondria
DNase)/ICAD (inhibitor of CAD) system
destroy
Cellular stress (growth factor withdrawal & p53
DNA
•
centres on
cell cycle arrest)
Regulated apoptosis is essential for
Bcl-2 family of ptns, Bax & Bak
expression of pro-apoptotic
tetrameric
Tissue structure formation in embryogenesis
14

15. 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
family member, tBid
pore complexes
pro-apoptotic
caspase 8 cleaves Bcl-2
formation of Bcl-2/Bax/Bak
if this complex is predominantly of
members
of
Bcl-2
family
apoptosome/caspase 9 & mitochondrial anti-IAPs
↑
apoptotic activation of effector caspases 3
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
membrane
imbed
to
outer
complex called apoptosome
initiator caspase (caspase 9)
caspase (caspase 3)
activates
activates effector
↓
✰ 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
permissive pores
binds
Conversely, overexpression of antiapoptotic Bcl-2
intrinsic & extrinsic apoptotic signalling
mitochondrial
Cytochrome C released from mitochondria
Apaf1
•
the cells
•
Therefore we must have nests of cells e’in all
different tissues that have not shut down their
genetic blueprint
15

16. •
These
stem
cells
give
rise
to
daughter
cell
(differentiated & limited ability to replicate) &
daughter cell w’ will not differentiate & has the
MOLECULAR BIOLOGY
infinite ability to replicate
•
In mammals
source categories of stem cells
Embryonic stem cells
Adult stem cells
found in adult tissues
Cord blood stem cells
•
DNA Structure & Function
derived from blastocysts
found in umbilical cord
The source of stem cells can also be subcategorized
by potency (specifies the potential to differentiate to
different cell types)
•
stranded DNA
•
guanine (G)) and pyrimidine (thymine (T) & cytosine
Produced from fusion of egg & sperm cell
(C)) bases of the nucleic acid
Produced by 1st few divisions of fertilized egg
differentiate
to
embryonic
&
extraembryonic cell types
Can differentiate to cells derived from any of
the 3 germ layers
Produce only cells of closely related family e.g.
RBCs, WBCs, etc.
Unipotent cells
•
of
self-renewal
distinguishes them from non-stem cells)
The monomeric unit in DNA (& RNA) is the nucleotide
(w’
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
•
Produce only 1 cell type
property
of base-pairs (bp)
•
Multipotent stem cells
haematopoietic stem cells
The length of DNA is generally measured in numbers
w’ is a base joined to sugar–phosphate unit
The descendants of totipotent cells
the
•
•
Pluripotent stem cells
Have
Each strand of DNA is made up of deoxyribose–
phosphate backbone & series of purine (adenine (A) &
Totipotent stem cells
Can
Genetic information is stored in form of double-
The large stretches of helical DNA are coiled around
histone ptns
nucleosomes & further condensed into
chromosomes that are seen at metaphase
16

17. •
In bacteria the coding sequences are continuous but in
higher organisms these coding sequences (exons) are
interrupted by intervening sequences that are noncoding (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
Genes
regulating cell
proliferation & apoptosis (in turn they are inactivated
by DNA methylation)
•
Gene is portion of DNA that contains codes for
polypeptide sequence
•
3 adjacent nucleotides (codon) code for particular aa
Transcription & Translation
e.g. AGA for arginine
•
Only 20 common aa but 64 possible codon combinations
make up genetic code
•
most aa encoded by >1 triplet
Conversion of genetic information to polypeptides &
ptns relies on transcription of sequences of bases in
Other codons used as signals for initiating or
terminating polypeptide-chain synthesis
•
•
DNA to mRNA
•
mRNA
Genes consist of lengths of DNA that contain
Found mainly in nucleolus & cytoplasm
sufficient
Polymers
nucleotide
triplets
to
code
for
the
of
nucleotides
containing
appropriate number of aa in polypeptide chains of
•
phosphate unit attached to base
particular ptn
The bases are A, G, C & uracil (U)
Genes vary greatly in size (most extend over 20–40
RNA
kbp) but few e.g. gene for muscle ptn dystrophin can
ribose–
complementary sequence ssDNA
is
ss
molecule
but
can
hybridize
e’
extend over millions of bp
17

18. •
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
modification
mRNA
post transcriptional
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)
•
•
Genetic information is carried from nucleus to
cytoplasm by mRNA
act as template for ptn
synthesis
•
Each
base
Alternative splicing is possible whereby entire exon
can be omitted
•
Processed mRNA
cytoplasm
in
mRNA
is
lined
up
opposite
corresponding base in DNA (C-G, G-C, U-A & A-T)
to
>1 ptn coded from same gene
migrates out of nucleus to
polysomes (groups of ribosomes) become
attached to mRNA
ribosomes consist of subunits
composed of small RNA molecules (rRNA) & ptns
18

19. •
rRNA components are key to binding & translation of
•
genetic code held by ribosomes & triplets of adjacent
bases on mRNA called codons are recognized by
For many genes, transcriptional control is the key
point of regulation
•
Deleterious (even oncogenic) changes to cell may arise
complementary sequences or anti codons in tRNA
through fault in expression of particular gene e.g. over
each tRNA molecule carries aa that is specific to anti
expression due to non-break down of mRNA
•
codon
•
As the ribosome passes along mRNA in the 5’-3’
direction (zipper linking)
aa transferred from tRNA
molecules & linked by ribosome
•
polypeptide chain
degradation (RNA interference, RNAi)
✰ Transcriptional control
•
st
Pathway that stops gene expression by ↑ RNA
Gene transcription (DNA to mRNA) is not spontaneous
1 20 or more nucleotides are recognition & regulatory
event
sequences
binding ptns (DBP) e’ genomic DNA
and
untranslated
but
necessary
for
translation
•
•
Translation begins when triplet AUG (methionine) is
•
Encountered
? only result of interaction of number of DNA
Regulation of gene expression must 1st start e’ opening
up of double helix of DNA in the correct region of Chr
all ptns start e’ methionine but it is
in order to do this
ptn molecules that recognize
often lost as the leading sequence of aa of native
•
the outside of DNA helix has evolved
peptides is removed during ptn folding
interact e’ major groove of DNA double helix
Similarly Poly A tail is not translated & is preceded by
stop codon
•
UAA, UAG or UGA
these DBP
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
The Control of gene expression
slight bend & if
repeated every 10 nucleotides it produces pronounced
curves
•
Gene expression is controlled at many points in steps
•
DBP that recognize these distorted helices
opening
between translation of DNA to ptns
•
up (or prevent opening) of the helix so the gene may
Ptns & RNA molecules are in constant state of
be transcribed
turnover
19

20. •
✰ Structural classes of DBP
•
Operator
Other DNA regulator ptns operate in close
4 basic DBP (according to structural motifs)
proximity to site of promoter binding (operator
Class of DBP
Helix–turn–helix
Zinc finger
Leucine zippers
Helix–loop–helix
ptns/regions & act either as repressors by binding
Examples
CREB (cAMP response element binding ptn)
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)
c-jun cell replication oncogene
c-fos cell replication oncogene
myc oncogene
mad oncogene
max oncogene
to DNA sequences e’in promoter site or as +ve
regulators facilitating RNA polymerase binding
•
Enhancer sequences are >200 bp away from site of
transcription initiation
Binding of regulator ptns to enhancer regions
(several
•
promoters, operators & enhancers
bases
from
promoter
site)
This turns out to be distance favourable for DNA
to loop back on itself e’out straining backbone
bonds of DNA double helix
DBP act as regulators of gene expression in 3
different ways
100
upregulates the expression
✰ Control regions & proteins
•
Enhancer
•
GAL4 enhancer of yeast
aid binding of transcription
factors to TATA region of promoter
Promoters
RNA polymerases bind to promoter region normally
general
adjacent to transcribed sequence of DNA
polymerase activity
•
transcription
factor
catalyst for
assembly
&
RNA
In mammals
cAMP response element (CRE) acts to↑
when number of DBP & DNA associated proteins
IC cAMP
activation & release of CREB
come together & interact (general transcription
transcription rate (but may also↓transcription)
In eukaryotes active transcription is possible only
factors)
these ptns thought to assemble at
•
↑
Repressors can↓transcription of gene by binding to
promoter sites used by RNA polymerases e.g. Pol II
regulatory sequence & blocking +ve regulators or by
that are characterized by specific motifs e.g.
interfering e’ promoter ptn assembly
TATA sequence
20

21. CHROMOSOMES, INTRONS & THE SIZE OF
HUMAN GENOME
•
•
enzymes
Coiling around histones & structural regions e.g.
centromeres & telomeres requires regions of DNA
Digestion of all cellular ptn by add of proteolytic
genomic DNA is isolated by chemical
extraction e’ phenol
•
DNA is stable & can be stored for years
devoted specifically to the purpose of packaging
•
10% of human DNA is highly repetitive (satellite DNA)
long arrays of tandem repeats
these regions tend
RESTRICTION ENZYMES & GEL
ELECTROPHORESIS
to be supercoiled around histones in condensed regions
Restriction enzymes cut dsDNA at specific sites
(heterochromatin)
•
•
•
Whenever human genomic DNA is cut e’ EcoRI
In contrast
most other DNA regions are relatively
restriction fragments (restriction fragment length
uncondensed (euchromatin)
•
The remaining DNA is either moderately repetitive
same
polymorphisms, RFLPs) are produced
•
As DNA is –ve charged molecule
genomic DNA
(30% of genome) or codes for unique genes (gene
fragments can be separated according to their size &
families occupying 2% of genome)
charge by electrophoresis through a gel matrix
DNA migrates to +ve anode & small fragments move
Tools for Molecular Biology
more quickly
•
DNA fragments separate out
Pulsed-field gel electrophoresis (PFGE) can be used to
separate very long pieces of DNA (100s of kilobases)
PREPARATION OF GENOMIC DNA
•
1st step in studying DNA of individual involves
preparation of genomic DNA
HYBRIDIZATION TECHNIQUES
•
When 2 strands separated(e.g. by heating)
they will
•
It is simple procedure in w’ any cellular tissue including
always re stick because of their complementary base
blood can be used
•
sequences
Cells are lysed in order to open their cell & nuclear
membranes
releasing chromosomal DNA
•
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
21

22. •
DNA probe is piece of ssDNA that can be labelled e’
radioactive isotope (usually
32
P) or fluorescent signal
•
Also called quantitative real time PCR (QRT-PCR)
•
will locate & bind to its complementary sequence
•
✰ Real-time PCR (RT-PCR)
Simultaneous quantification & amplification of given
Hybridization is exploited in number of techniques
including
DNA sequence
•
Southern blot
DNA fragments separated by gel
sequence is present in sample e.g. viral genome & if
electrophoresis & transferred onto membrane
sheet
Northern
present, the number of copies in the sample
•
blot
RNA
by
RT-PCR is combined e’ reverse transcription PCR to
gel
quantify low abundance mRNA enabling researcher to
electrophoresis & transferred onto membrane
quantify relative gene expression at particular time in
sheet
particular cell/tissue
In situ hybridization
separated
It can be used to determine whether specific
localization of native
nucleic acid sequences e’in the cell & its component
organelles, including chromosomes
✰ Expression microarrays/gene chips
•
abundance of mRNA for 1000s of genes present in
cells/tissue of different types e.g. to examine changes
THE POLYMERASE CHAIN REACTION (PCR)
•
•
in gene expression from normal tissue to that of
Minute amounts of DNA can be amplified over million
times e’in few hours
The technique has 3 steps
malignant colonic polyps
•
different regions of known genes onto solid surface in
Then cooled to favour DNA annealing & primers
Finally
DNA polymerase extend the primers in
opposite directions using target DNA as template
•
After one cycle
4 copies
2 copies of dsDNA, after 2 cycles
The basic technology is the ability to immobilize
sequences of DNA complementary to specific genes or
ds genomic DNA is denatured by heat into ssDNA
bind to their target DNA
It is methodology developed to examine relative
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
22

23. to their complementary microdot & detected by laser-
reasonably large sequences as plasmids e’in host
induced excitation of fluorescent tag
bacteria
position, light
trick bacteriophages in packaging them to
wavelength & intensity recorded by scanning confocal
viral body & this viral body is then able to infect
microscope
target bacteria
relative intensity of Cy5-red : Cy3-
green is reliable measure of relative abundance of
•
specific mRNAs in each sample
Yellow
Red
equal binding of both fluorescent tagged
cloning
& creates many copies of recombinant DNA molecule
(in vivo)
•
no hybridization
overexpression
Green
•
DNA fragment of interest is inserted in the vector
DNA sequence using enzyme ligase (in vitro)
mRNA
Black
efficient transfection rates
from mRNA sequence by reverse transcriptase enzyme
under expression
Power of the system
Alternatively it could be cDNA w’ has been copied
ssDNA
many 1000s of genes screened
DNA polymerase
dsDNA contains all
sequences necessary for functional gene but unlike
for expression & relative expression in normal &
genomic DNA it lacks introns
diseased tissue
The Biology of Chromosomes
DNA CLONING
•
Particular DNA fragment of interest isolated &
inserted to genome of simple self replicating organism
HUMAN CHROMOSOMES
or organelles e.g. viruses & plasmids
•
Each diploid cell nucleus contain 6×109 DNA bp in Chr
•
Vectors include
•
Chromosomes contain one linear molecule of DNA
•
Each vector takes optimum size of cloned DNA insert
bacteriophage viruses; plasmids
(viruses accommodate only small sequences, larger
wounded around histone in small units (nucleosomes)
•
Diploid
human
cells
have
46
chromosomes
(23
fragments can be inserted in plasmid & larger in yast
Chr)
•
inherited from each parent)
22 pairs of autosomes + 2 sex chromosomes(XY/XX)
Hybrid between plasmid & bacteriophage (cosmid)
constructed
artificially
&
has
ability
to
clone
•
23 homologous pairs
Chromosomes classified according to their size &
shape (the largest is Chr 1)
23

24. •
The constriction in Chr is centromere
metacentric
(in middle of Chr) or acrocentric (at one extreme end)
•
THE X CHROMOSOME & INACTIVATION
•
1 of 2 X Chr in cells of ♀ becomes transcriptionally
Centromere divides Chr into short arm (p) & long arm
inactive
(q)
inactivation or Lyonization phenomenon)
e.g. CFTR gene (of cystic fibrosis) maps to 7q21
on Chr 7 in long arm in band 21
•
•
Indications for chromosomal analysis
cell has only 1 dose of X-linked genes (X
Inactivation is random & can affect
either
X
chromosome
Antenatal
Pregnancies in women >35 years
+ve maternal serum screening for aneuploid
TELOMERES & IMMORTALITY
•
pregnancy
U/S features consistent e’ aneuploid fetus
Ends of Chr (telomeres) do not contain genes but many
repeats of hexameric sequence TTAGGG
•
Replication of linear Chr start at coding sites (origins
Severe fetal growth retardation
of replication) e’in main body of Chr (not at 2 extreme
Sexing of fetus in X-linked disorders
ends)
In the neonate
•
Extreme ends are susceptible to ssDNA degradation
Congenital malformations
back to dsDNA
Suspicion of trisomy or monosomy
consequence of multiple rounds of replication e’
Ambiguous genitalia
consequential telomere shortening
In the adolescent
cellular ageing measured as genetic
Chr instability &
cell death
1ry amenorrhoea or puberty development failure
•
Stem cells have longer telomeres > daughter
Growth retardation
•
Germ
In the adult
cells
replicate
e’out
shortening
of
their
telomeres because they express enzyme telomerase
Screening parents of child e’ chromosomal
(protects against telomere shortening by acting as
abnormality for further genetic counselling
template primer at extreme ends of Chr)
Infertility or recurrent miscarriages
•
Learning difficulties
Certain malignant disorders e.g. leukaemias &
Wilms’ tumour
Most somatic cells (unlike germ & embryonic cells)
switch off activity of telomerase after birth
•
Many cancer cells reactivate telomerase contributing
to their immortality
24

25. THE MITOCHONDRIAL CHROMOSOME
•
In addition to 23 pairs of Chr in nucleus, mitochondria
GENETIC DISORDERS
in cytoplasm have their own genome
•
Mitochondrial Chr is circular DNA (mtDNA)
Approximately 16’500 bp
•
Every bp make up part of coding sequence (no
Spectrum of inherited or congenital genetic disorders
classified as
introns)
Chromosomal
Principally encode ptns or RNA molecules involved
chromosome disorders
in
The Mendelian disorders
mitochondrial
function
(components
of
mitochondrial respiratory chain)
•
Every
cell
contain
mitochondrial Chr
100s
mitochondria
•
100s
mitochondrial
Variety of non-Mendelian disorders & multifactorial
disorders all are result of mutation in genetic code
virtually all mitochondria are
inherited from mother (sperm head contain no or few
mitochondria)
including
Sex-linked single-gene disorders
Critical role in apoptotic cell death
•
disorders,
Chromosomal disorders
•
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
25

26. •
If non-disjunction occurs during meiosis
ovum or
sperm e’ either
Extra Chr
No Chr
trisomy (3 instead of 2 copies of Chr)
monosomy (1 instead of 2 copies of Chr)
Examples
Only trisomy 13, 18 & 21 (Down’s syndrome)
Deletion
Duplication
Inversion
survive to birth (most children e’ trisomy 13 &
Balanced translocation
18 die in early childhood)
Full autosomal monosomies
extremely rare &
very deleterious
Sex Chr trisomies e.g. Klinefelter’s syndrome
(44+XXY) are relatively common
Sex Chr monosomy e.g. Turner’s syndrome
(44+X0)
•
copy remaining on the non-deleted homologous)
Occasionally non-disjunction during mitosis
after 2 gametes fused
Example
2 cell lines each e’ different
Chr complement (more often e’ sex Chr)
•
shortly
Prader Willi syndrome
mosaicism
Very rarely
deletion of part long arm of Chr 15
entire chromosome set will be present
in >2 copies
triploidy (69 Chr) or tetraploidy (92
Chr)
cytogenetic events
Wilms’ tumour
deletion of part of short arm
of Chr 11
spontaneous abortion
DiGeorge syndrome
microdeletions in long
arm of Chr 22
ABNORMAL CHROMOSOME STRUCTURES
•
Duplications
•
Abnormal Chr structures can disrupt DNA & genes
When portion of Chr is present on the Chr in 2
•
Deletions
copies
Deletions of portion of Chr
genes in that Chr portion are present in
disease if 2 copies
extra dose e.g. Charcot–Marie–Tooth disease (form
of genes in deleted region are necessary (the
of neuropathy) is due to small duplication of region
individual will not be normal e’ the 1 copy remaining)
of Chr 17
26

27. •
Inversion
o Clinically relevant is 14/21 Robertsonian
End to end reversal of segment e’in a chromosome
e.g. abcdefgh becomes abcfedgh (haemophilia)
•
translocation in woman
having baby e’ Down’s syndrome (male carrier
Translocations
1 in 8 risk of
has 1 in 50 risk)
2 Chr regions join together (not normally do)
Chr translocations in somatic cells
o 50%
tumorigenesis
risk
of
themselves
Translocations can be very complex involving >2 Chr
producing
genetic
carrier
family
study
like
is
necessary
but most are simple & fall in 1 of 2 categories
Reciprocal translocation
o When any 2 non homologous Chr break
simultaneously & rejoin, swapping ends
MITOCHONDRIAL CHROMOSOME
DISORDERS
•
No introns in mitochondrial genes
mutation has high
o Cell still has 46 Chr (2 of them rearranged)
chance of having effect however as every cell contains
o Someone e’ balanced translocation is likely to
100s of mitochondria so single altered mitochondrial
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
genome is not noticed
Chr
&
have
unbalanced
translocation e’ physical manifestations
As mitochondria divide
mitochondria
•
Most
↑likelihood of more mutated
mitochondrial disease
mitochondrial
diseases
are
myopathies
&
neuropathies e’ maternal pattern of inheritance
Myopathies
(CPEO) chronic progressive external
Robertsonian translocation
ophthalmoplegia
o When 2 acrocentric Chr join & short arm is
Encephalomyopathies
lost
only 45 Chr
(MERRF)
myoclonic
epilepsy with ragged red fibres
o It is balanced translocation as no genetic
MELAS
mitochondrial encephalomyopathy, lactic
material is lost & the individual is healthy but
acidosis & stroke-like episodes
any
Kearns–Sayre syndrome
offspring
have
risk
of
inheriting
ophthalmoplegia, heart
unbalanced arrangement depending on w’
block,
acrocentric Chr is involved
deficiency due to long deletions & rearrangements
cerebellar
ataxia,
deafness
&
mental
27

28. 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
Gene Defects
•
Mendelian & sex-linked single-gene disorders are due
to mutations in coding sequences & their control
elements
(LHON) Leber’s hereditary optic neuropathy
•
All cause dysfunction of the protein product
commonest cause of blindness in young men e’
bilateral
loss
arrhythmias
of
central
vision
&
cardiac
it is mitochondrial disease caused
by point mutation in one gene
Multisystem
(sideroblastic
disorders
anaemia,
MUTATIONS
✰ Point mutation (Missense mutation)
•
Pearson’s
pancytopenia,
syndrome
exocrine
The simplest type of change
•
Substitution of 1 nucleotide for another
change
codon in coding sequence
28

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
Missense mutation
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
Nonsense mutation
✰ Insertion or deletion
•
Insertion or deletion of 1 or more bases is more
serious as it
•
examples
alteration of rest of the following
Large deletions in dystrophin gene remove coding
sequence (frame-shift mutation)
•
sequences
Example
genotypes II, ID & DD
TAA’GGA’GAG’TTT
TA-G’GAG’AGT’TT
In both cases
different aa incorporated in
polypeptide chain
•
It is responsible for some forms of thalassaemia
deletion of 287 bp
repeat sequence & DD is associated e’ higher
Extra nucleotide (A) is inserted
If 3rd nucleotide (A) is deleted
Duchenne muscular dystrophy
Insertion/deletion (ID) polymorphism in ACE gene
If the original code was
TAA’AGG’AGA’GTT’T
Insertions & deletions can involve 100s of bp of DNA
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
29

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
where instead of ‘stop’ sequence
Hb variant
single base change
insertion of extra aa
SINGLE-GENE DISEASE
•
Monogenetic disorders involving single genes can be
inherited as dominant, recessive or sex-linked
•
•
Estimation of risk to offspring for counselling families
can be difficult because
Many syndromes show multiple forms of inheritance
pattern because multiple defects occur in given
Great
disease associated gene or in separate genes for
incomplete penetrance
example in Ehlers–Danlos syndrome
disorder but does
AD, AR & XL
Variable
✰ Autosomal dominant disorders (AD)
•
•
AD disorder occurs when 1 of 2 copies of autosomal
if patients have dominant
not manifest
clinically
due to new mutation
Offspring of heterozygotes
50% inheriting Chr
also have the disease
dominant
traits
are
risk of further affected
child is negligible e.g most cases of achondroplasia
Heterozygous individual e’ 2 different forms (or
manifest the disease
expression
New cases in previously unaffected family may be
cannot compensate
carrying disease allele
manifestation
parent may have severely affected child
Chr has mutation & ptn produced by normal gene
•
their
extremely variable in severity e.g. mildly affected
Overall incidence 7 in 1000 live births
alleles) of same gene
in
appearance of the gene having skipped generation
inheritance
•
variability
are due to new mutations
✰ Autosomal recessive disorders (AR)
•
Overall incidence 2.5 in 1000 live births
30

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
31

32. Affected males tend to have severe disease >
heterozygous female
•
Only males are however
linked single gene disorders
•
X linked recessive disorders (XLR)
no known examples of Y
Sex-limited inheritance
These disorders present in males & homozygous
Occasionally a gene can be carried on an autosome
female (usually rare)
but manifest only in one sex
Transmitted
by
healthy
female
carriers
or
an AD in males but behave as AR in females
✰ Other single-gene disorders
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
one break point e’in intron 22 of
factor VIII gene)
These are disorders w’ may be due to mutations in
single genes but do not manifest as simple monogenic
intra Chr rearrangement (inversion) of tip of long
arm X-Chr
frontal baldness is
disorders
•
They can arise from variety of mechanisms
Triplet repeat mutations
Offspring of carrier female + normal male
50% of girls are carriers
In gene responsible for dystrophia myotonica
inherit mutant allele
mutated allele was found to have expanded
from their mother & normal allele from their
3’UTR region in w’ three nucleotides (CTG) was
father
repeated up to 200 times
50% of girls
inherit 2 normal alleles
50% of boys
have haemophilia as they inherit
late onset disease had 20–40 copies of the
mutant allele from their mother (& Y Chr from
repeat but their children & grandchildren who
their father)
presented e’ disease from birth
50% of boys are normal
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
In families e’ dystrophia myotonica
people e’
had increase
in number of repeats (up to 2000 copies)
number of triplets affects mRNA & ptn function
ً ‫ﺗﻡ ﻣﻧﺎﻗﺷﺗﻪ ﺳﺎﺑﻘﺎ‬
Mitochondrial disease
Imprinting
In some way (not yet clear), the fetus can
distinguish between Chr inherited from mother
32

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
Measurements of most biological traits e.g. height is
Deletion of part of long arm of Chr 15 (15q11–
variant thought to be due to additive effects of
q13)
number of alleles at number of loci many of w’ are
Prader–Willi syndrome if it is paternally
inherited but deletion of similar region of the
individually
Chr
techniques
Angelman’s syndrome if it is maternally
•
inherited
identified
using
molecular
biological
There are sex differences e.g. congenital pyloric
The affected gene is identified as ubiquitin
stenosis is most common in boys but if it occurs in
(UBE3A)
girls
Significantly
maternal Chr 15 UBE3A is
expressed in brain & hypothalamus
•
defective
larger number of affected relatives
Most human diseases e.g. heart disease, DM and
common mental disorders are multifactorial traits
maternal ubiquitin in Angelman’s syndrome
accumulation of undegraded ptn & neuronal
damage
COMPLEX TRAITS: MULTIFACTORIAL &
POLYGENIC INHERITANCE
Genetic Counselling
•
Aims of genetic counselling
Obtain full history
pregnancy history, drug,
•
Combination of genetic & environmental factors are
alcohol ingestion during pregnancy & maternal
said to be multifactorial
•
illnesses
Those involving multiple genes are said to be polygenic
Establishing
accurate
diagnosis
of
genetically
abnormal child
33

34. Draw family tree & questions about abortions,
All serum marker are corrected for gestational
stillbirths, deaths, marriages, consanguinity
ages
Estimate risk of future pregnancy being affected
the appropriate gestation week is necessary
multiple of the mean (MOM) value for
Give information about prognosis & management
Chorionic villus sampling (CVS) at 11–13 weeks under
Continued support & follow-up
U/S control to sample placental site
Genetic screening including prenatal diagnosis
Amniocentesis at 15 weeks to sample amniotic fluid
•
The triple test for Chr abnormalities
PRENATAL DIAGNOSIS
•
α-fetoprotein (low)
Human
7–11 Weeks
chorionic
gonadotrophin
(high)
for
Down’s syndrome & neural tube defects
Vaginal U/S
The quadruple test
Confirm viability, fetal number & gestation by
The triple test + inhibin-A ( ↑ in Down’s
crown rump measurement
syndrome)
11–13 Weeks & 6 days (combined test)
If too late for triple test or previous option not
U/S for nuchal translucency measurement (normal
fold <6 mm)
↑in neural tube defects
Unconjugated oestradiol (low)
✰ Investigations depend on gestation
•
testing
maternal serum for
Should be offered to all pregnant women in UK but it
is offered to high risk mothers only
•
14–20 Weeks (serum triple or quadruple test)
detect major Chr abnormalities e.g.
trisomies & Turner’s syndrome
Maternal serum is tested for
PAPP-A (pregnancy associated plasma protein-A)
from syncytial trophoblast
β-HCG for trisomy 21
Combined test is more accurate > triple test
alone at 16 weeks
offered
•
14–22 Weeks
U/S for structural abnormalities e.g. neural tube
defects, gestation period
The best time to detect congenital heart defects
is 18–22 weeks
Reported detection rates for all congenital defects
vary from 14 to 61% for hypoplastic ventricle to
97-100% for anencephaly
34

35. Mutation
Gene Therapy
o The commonest is single mutation e’ 3 bp
deletion in exon 10
•
removal of codon
specifying phenylalanine (F508del)
Gene therapy entails placing normal copy of gene into
the cells of patient who has defective copy of the
o Also >1000 different minor mutations of
gene (concentrating on recessive disorders e.g. cystic
CFTR gene e’ most mapping to ATP-binding
fibrosis where the disease is due to absence of normal
domains
gene product)
Gene therapy experiments
•
In dominant disorders it is difficult & complicated
o
•
2 major factors are involved in gene therapy
Still under trial to restore CFTR function by
transfection of cells e’ wild type receptor
Introduction of functional gene sequence in target
o 2 different routes are tried
cells
♦ Placing CFTR gene in adenovirus vector
Expression & permanent integration of transfected
♦ Placing CFTR gene in liposome (conveyed
to lung by aerosol spray)
gene in host cell genome
•
of liposome fuses e’ cell membrane to
Suitable diseases for current gene therapy include
deliver CFTR DNA into cell
Cystic fibrosis
CFTR gene
o Cystic
fatty surface
o Topical nasal gentamicin (aminoglycoside AB)
fibrosis
transmembrane
gene is the responsible for cystic fibrosis
o It was 1
st
expression of functional CFTR channels
regulator
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
Adenosine deaminase (ADA) deficiency
Rare immunodeficiency disease
normal
human
lymphocytes
&
humoral
ADA
gene
introducing
in
patient’s
reconstitute function of cellular
immunity
in
severe
combined
immunodeficiency
Familial hypercholesterolaemia
It is due to↓LDL receptor gene
channel
35

36. Gene therapy
receptor gene is inserted in
hepatocytes (removed by liver biopsy)
gene-
corrected hepatocytes
back
•
reinjected in portal
circulation
✰ Cancer
migrate
to
reincorporated
start
to
produce
receptor protein
LDL
dramatically ↓ cholesterol
genetic
p53 is TSG
disease
&
many
genes
are
apoptosis in cells e’ damaged genetic
material
reintroduction
&
overexpression
of
functional p53 in tumours is investigated
•
level
is
deregulated
•
liver
Cancer
Since it is only likely to occur in rapidly dividing cells
perfect target for cancer gene therapy by repeat
TREATMENT OF SOMATIC DISEASE
exposure to vectors e.g. retroviruses, liposomes &
✰ Vascular disease
•
naked DNA plasmids
Neovascularization to ↑ blood 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
Tumour growth depends on development of new blood
vessels (angiogenesis) & inhibitors are under trial
✰ Stem cell therapy
•
repair damaged
& diseased arteries
•
•
Number of adult stem cell therapies already exist
particularly bone marrow transplants
•
It is anticipated to treat wide variety of diseases
Deliver liposomes loaded e’ DNA or direct inject of
require
DNA plasmids to tissue
Parkinson’s, spinal cord injuries & muscle damage
ptn will be expressed by
cells (only 1–3% but it is sufficient for local effect
•
required)
destroyed
tissues
e.g.
The blood in umbilical cord is available & rich source of
colonize bone marrow & rapidly populating marrow e’ all
Neurotrophic factors can be transiently expressed
same as e’ vascular diseases
of
haemopoietic stem cells i.e. CD34 +ve & CD38 –ve
✰ Neuronal disease
•
replacement
nerve cell regeneration
various cells (RBC’s & WBC’s)
•
Umbilical cord stem cell, dubbed cord blood-derived
& maintenance
•
embryonic
Extend expression period of neurotrophin by injecting
differentiate to more types of tissue not simply
transfected myocytes in damaged area
haemopoietic cells (super pluripotentiality)
fuse e’ any
like
stem
cells
(CBEs)
able
to
adjacent muscle
36

37. •
Primitive monocyte derived multipotential cell (MOMC)
could be isolated from adult peripheral circulating
monocytes
induced (given the correct paracrine,
environmental
& adhesion signals)
endothelia,
•
Cancers are genetic diseases & involve changes to
normal function of cellular genes
neurones, cardiomyocytes & mesenchymal lineages
•
The Genetic basis of Cancer
Similar reports concerning adult stem cells isolated
•
Multiple genes interact during oncogenesis & stepwise
progression of defects leads over proliferative of
from skin
particular cell to full breakdown of control ( apoptosis)
•
THE HUMAN PROTEOME PROJECT
•
Studying of ptn expression characteristics of normal
& diseased cells
•
Pattern
of
expressed
dots
corresponds
to
different
cancer can be inherited
•
Cancer tissues are clonal & arise from changes in only
one cell w’ then proliferates in the body
Achieved by using 2D gel electrophoresis
•
Susceptibility to development of particular form of
ptn
•
changes w’ lead to cancer fall in 2 categories:
non-, over- & underexpression of given
ptn can be detected by corresponding change on
proteome
The genes that are primarily damaged by genetic
oncogenes & TSG
•
Oncogenesis is multistep process
number of
Post-translational modifications of ptns show up as
mutations or alterations to key genes are required
change in either size or charge on proteome picture
•
before malignant phenotype is expressed
•
Once mutations begun to cause unchecked clonal
expansion of 1ry tumour cells
further mutations
occur e’in subsequent generations of daughter cells
clones w’ are invasive & or form metastases
ONCOGENES
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)
•
Genes coding for growth factors, growth factor
receptors, 2ry messengers or even DBP would act as
promoters of abnormal cell growth if mutated
37

38. •
By chance some of these point mutations will
promote oncogenesis (v-oncogenes) & later their
occur in regions of oncogene
normal cellular counterparts (c-oncogenes) were found
that gene
Oncogenes encode ptns that participate in regulation
Not all bases in oncogene cause cancer if
of
•
Viruses carry genes w’ when integrated to host cell
Mutated but some do (those in coding region)
normal
cellular
proliferation
e.g.
erb-A
on
chromosome 17q11–q12 encodes for thyroid hormone
receptor
② Chromosomal translocation
If during cell division an error occurs & 2 Chr
translocate
Examples of acquired/somatic mutations
& proto-oncogenes
Pancreatic cancer
DNA amplification
Myc
HER2-neu
Neuroblastoma
Breast cancer
Chromosome translocation
BCR-ABL
PML-RAR
Bcl-2/IgH
c-myc & Ig
CML, ALL
APML
Follicular lymphoma
Burkitt’s lymphoma
Example of fusion gene (Philadelphia Chr) in
GML
Similarly in Burkitt’s lymphoma
oncogene by regulatory segment of unrelated Ig
Transformation to oncogenes can occur by 3 routes
① Mutation
cigarette
smoke,
ionizing
radiation UVR can cause point mutation in
genomic DNA
translocation
replace the regulatory segment of myc
(proto-oncogenes)
e.g.
gene are
inappropriately
Non activated oncogenes w’ are functioning normally
Carcinogens
over
end of 1 gene is translocated
sequences of 1 part of fusion
✰ Activation of oncogenes
•
swaps
on to beginning of another gene (fusion gene)
CML, chronic myeloid leukaemia; ALL, acute lymphoblastic
leukaemia; APML, acute promyelocytic leukaemia
•
portion
translocation breakpoint in middle of 2 genes
If this happens
Point mutation
K-ras
activation of
③ 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
38

39. sarcoma virus of chickens was found to induce
The ptn encoded by p53 is cellular 53 kDa nuclear
cancer because it carried ras oncogene
phosphoprotein (plays role in DNA
After the initial activation other changes occur
synthesis in control of cell cycle, differentiation &
in DNA
apoptosis)
repair &
p53 is DBP
TUMOUR SUPPRESSOR GENES (TSG)
•
Activate many gene expression pathways but it
is normally only short lived
contrast to oncogenes) & induce repair or self
p53 is likely to act as tetramer
destruction (apoptosis) of cells contain damaged DNA
single
Example
germline mutations in genes found in non-
formation because hetero tetramer of mutated
polyposis
•
These genes restrict undue cell proliferation (in
CRC
&
responsible
for
repairing
DNA
1st TSG to be described was RB gene
RB
normal
of
p53
gene
can
subunits
promote
would
tumour
still
be
dysfunctional
mismatches
•
copy
mutation in
In many tumours
mutations in
function
Retinoblastoma
also prevent its cellular catabolism
although in some cancers there is loss of p53 from
1 in 20’000 young children
Familial variety of retinoblastoma
mutations that disable p53
1st mutation is
both Chr in most cancers (particularly CRC)
such
inherited & by chance 2nd somatic mutation occurs
long lived mutant p53 alleles can disrupt normal
e’ the formation of tumour
alleles ptn
Sporadic variety of retinoblastoma
by chance
How TSG work?
both mutations occur in both RB genes in a single
TSG products are involved in control of cell cycle
cell
•
•
•
Progression through cell cycle is controlled by many
Other TSG
molecular gateways w’ are opened or blocked by cyclin
gene p53
Mutations in p53 have been found in almost all
group of ptns that are specifically expressed at
human tumours including sporadic CRC, carcinomas
various stages of the cycle
of breast & lung, brain tumours, osteosarcomas &
leukaemias
•
RB & p53 proteins control cell cycle & interact
specifically e’ many cyclin ptns (The latter are
affected by INK 4α acting on p16 ptns)
39

40. •
General principle
being held at 1 of these gateways
of high variability in repeat number between
programmed cell death
•
p53
induces expression of other genes & its own
expression is induced by broken DNA
Often used as markers for linkage analysis because
initially cause
individuals
These regions are inherently unstable & susceptible
expression of DNA repair enzymes, if repair is too
to mutations
slow or cannot be effected then other ptns induced by
Somatic microsatellite instability (MSI) has been
p53 will effect programmed cell death
detected in number of tumours
✰ Viral inactivation of tumour suppressors
Detecting
MSI
involve
comparing
length
of
Suppression of normal TSG function by disabling
microsatellite alleles amplified from tumour DNA e’
normal ptn (once it is transcribed) rather than by
the corresponding allele in normal tissue from same
mutating the gene
individual
Viruses have developed their own genes w’ produce
Recent studies indicate that MSI can be detected
ptns to do precisely this
in 90% of tumours from individuals e’ hereditary
The main targets of these ptns are RB & p53 to w’
non-polyposis CRC
they bind & disable
The presence of these additional microsatellite
Adenovirus E1A & HPV E7 gene products bind RB
alleles (repeated segments) in tumour cells results
Adenovirus E1B & HPV E6 gene products bind p53
from inherent susceptibility of these areas to such
SV40 virus large T Ag binds both RB & p53
alterations & from mutations in DNA mismatch
repair mechanism that would normally correct
✰ Microsatellite instability
Microsatellites are short (50–300 bp) sequences
composed of tandemly repeated segments of DNA
these errors
✰ Tumour angiogenesis
2-5 nucleotides in length (di/tri/tetranucleotide
Once a nest of cancer cells reaches 1–2 mm in
repeats) scattered throughout the genome in non-
diameter
coding regions between genes or e’in genes (introns)
survive & grow as diffusion is no longer adequate to
Many
supply the cells e’ O2 & nutrients
of
these
microsatellites
are
highly
it must develop blood supply in order to
polymorphic
40

41. 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 e’out
harming them
‫ﺗﻣﺕ ﺑﺣﻣﺩ ﷲ ﻭﻓﺿﻠﻪ‬
41