Basic Genetics for MRCP
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Basic Genetics for MRCP part 1

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Basic Genetics for MRCP Basic Genetics for MRCP Presentation Transcript

  • 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
  • • 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
  • • 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
  • • 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
  • • 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
  • • 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
  • 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
  • • 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
  • 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
  • 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
  • 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
  • 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
  • • • 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
  • • 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
  • 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
  • • 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
  • • 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
  • • 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
  • • 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
  • • ✰ 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
  • 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
  • • 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
  • 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
  • • 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
  • 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
  • • 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
  • • 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
  • 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
  • • 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
  • ✰ 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
  • • 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
  • 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
  • & 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
  • 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
  • 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
  • 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
  • • 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
  • • 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
  • 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
  • • 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
  • 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