Med Bioquimica Examen 1 Repaso
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Organization*of*the*Human*Genome*
• Human&Chromosome*o Chromatin/&Complex&of&DNA&and&protein*o 44&homologous&chromosomes&and&2&sex&chromosomes*o Complementary&DNA&with&different&dyes*o Karyotype/&Arrangement&of&the&full&chromosome&set*o *Giesma&Staining*
• Human&Genome&Composition*o The&human&genome&comprises&two&parts*
! A&complex&nuclear&genome*• Provides&the&bulk&of&genetic&information*• Partitioned&in&22&autosomes&plus&an&X&chromosome&in&females&and&an&
additional&Y&chromosome&in&males*! A&simple&mitochondrial&genome*
• Only&37&genes*• A&single&type&of&small&circular&DNA*
o There&are&overlapping&genes&as&well&as&gene/within/genes.&Furthermore,&there&are&pseudogenes&and&retrogenes,&which&originate&from&reverse&transcription&from&RNA&transcripts*
o Diseases&such&as&Fragile&X&syndrome&and&Huntington&disease&are&associated&with&the&expansion&of&nucleotide&triplet&repeats,&a&phenomenon&which&may&result&from&slipped&mispairing&during&DNA&synthesis.*
• DNA&Supercoiling*o There&are&several&levels&of&organization,&one&of&which&is&the&supercoiling&of&the&double/
stranded&DNA&helix*o DNA&in&the&cell&must&be&organized&to&allow:*
! Packing&of&large&DNA&molecules&within&the&cells*! Access&of&proteins&to&read&the&information&in&DNA&sequence*
o Normal&plasmid&DNA&in&bacteria&is&also&usually&supercoiled*! Comes&in&two&flavors&(Right/handed&negative&superhelix&Lk&=&/2&and&Left/handed&
positive&superhelix&Lk&=&+2)*o Linking&number&in&relaxed&DNA*
! Number&of&base&pairs÷d&by&the&number&of&base&pairs&per&helical&turn*! Topoisomers&differ&by&Linking&Number*! *Compacted&negatively&supercoiled&DNA&travels&fastest*
• Topoisomerase*o Enzymes&that&change&linking&number&of&DNA*o Required&for&DNA&unwinding&and&rewinding&during&transcription&and&replication*o Two&major&types:*
! Type&I/&Makes&cut&in&one&DNA&strand;&linking&number&increases&by&one*! Type&II/&Makes&cut&in&both&DNA&strands;&linking&number&increases&by&two*
o In&bacteria,&HU&bends&DNA&sharply&into&a&tight&circle&and&is&primarily&responsible&for&the&formation&of&a&beaded&nucleoid&structure&(has&40&loops,&unique&to&chromosome&packing&in&prokaryotes)*
• Nucleosome/&Basic&level&of&chromosome/chromatin&organization*• Histone*
o DNA&binding&proteins&(a&Chromatin?)*o Highly&basic*o Positive&charge&(Arg&and&Lys)*o Forms&an&octamere*o Core&histones&are&highly&conserved&proteins,&sharing&a&structural&motif&called&histone&
fold*o H1&is&particularly&important&(?)*o Exposed&to&covalent&modifications*
! Acetylation&of&lysines&(promotes)*! Methylation&of&lysines&(inhibits)*! Phosphorylation&of&serines&(CHECK&WHAT&IT&DOES)*
o Once&wrapped&with&DNA,&it&has&one&net&negative&supercoil*• Epigenetics/&The&study&of&heritable&changes&in&gene&function&that&occur&without&a&changing&DNA&
sequence*o Used&in®ulation&of:*
! Correct&organization&of&chromatin*! Genomic&imprinting*! Silencing&of&repetitive&elements*! X&chromosome&inactivation*
&
*
DNA*Replication*
• DNA&replication&o Process&by&which&the&genetic&material&is&copied&o Original&DNA&strands&used&as&templates&o Relies&on&the&complementarity&of&DNA&strands&
! AT/GC&rule&(Chargaff’s&rule)&o Process:&
! Two&DNA&strands&come&apart&! Each&serve&as&template&for&new&strand&! Two&daughter&strands&and&two&parental&strands&
o Semiconservative&model&• Bacterial&DNA&replication&
o Synthesis&begins&at&Origin&(oriC)&! Only&one&origin&per&chromosome&
o Bidirectional&o Replication&forks&meet,&ending&replication&
o Three&important&types&of&DNA&sequences&in&oriC&! AT/rich®ion&
• HU&binds,&causing&the®ion&to&wrap&around&the&DnaA&protein&and&separates&the&AT/rich®ion&
! DnaA&boxes&• DnaA&proteins&bind&to&the&DnaA&box,&stimulating&the&cooperative&
binding&of&additional&DnaA&proteins&to&form&a&complex&! GATC&methylation&sites&
o DNA&polymerases&! Five&in&bacteria:&
• DNA&pol&I&and&III/&Normal&replication&o pol&I&
! Single&polypeptide&! Removes&RNA&primers&and&replaces&them&with&DNA&! 5’/>3’&exonuclease&activity&to&digest&RNA&! 5’/>3’&polymerase&activity&to&replace&it&with&DNA&! Gap&made,&filled&by&DNA&ligase&
o pol&III&! 10&subunits&(holoenzyme)&! Remains&attached&(processive)&! α&synthesizes&DNA&! β&is&clamp&! γ&is&clamp/loader&
• DNA&pol&II,&IV&and&V/&DNA&repair&o Replication&process:&
! DNA&helicase&separates&the&two&DNA&strands&by&breaking&the&hydrogen&bonds&between&them&(generates&positive&supercoil,&fixed&by&DNA&gyrase)&
! Single/stranded&binding&proteins&bind&to&the&separated&DNA&strands,&keeping&them&apart&
! Short&RNA&primers&are&synthesized&by&DNA&primase&! DNA&pols&attach,&creating&two&new&daughter&strands&(always&5’/>3’)&
• Leading&strand&o One&RNA&primer&made&at&origin&o DNA&pol&III&
• Lagging&strand&o Looped&o Many&RNA&primers&required&o DNA&pol&III&synthesizes&small&DNA&fragments&(Okazaki&
fragments)&! Termination&of&Replication&
• ter&sequences&(T1&and&T2)/&Termination&sequences&opposite&to&oriC&• tus&protein&binds&to&ter,&stopping&movement&of&replication&forks&• When&forks&meet,&DNA&ligase&covalently&links&all&four&DNA&strands,&
resulting&in&two&interwinded&molecules&(now&called&catenanes)&
• Catenanes&are&separated&by&topoisomerases&o DNA&Replication&Complexes&
! Primosome&=&DNA&Helicase&+&Primase&! Replisome&=&Primosome&+&DNA&polymerase&holoenzyme&! Dimeric&DNA&polymerase&=&Two&DNA&pol&III&proteins&act&in&concert&to&replicate&
both&the&leading&and&lagging&strand&o Proofreading&Mechanisms&
! DNA&replication&has&high&fidelity&thanks&to:&• Instability&of&mismatched&pairs&• Configuration&of&the&DNA&polymerase&active&site&(induced&ift)&• Proofreading&function&of&DNA&polymerase&3’/>5’&exonuclease&activity&
o Bacterial&DNA&replication&is&coordinated&with&cell&division&o Methylation&is&used&to&facilitate&initiation&of&DNA&replication&
*
Eukaryotic*DNA*Replication*
• Multiple&origins&of&replication&• Bidirectional&• ARS&elements&(Autonomnously&Replicating&Sequences)&
o Found&at&origins&o High&%&of&A&and&T&o Similar&to&bacterial&DnaA&boxes&
• Origin&Recognition&Complex&(ORC)&o A&six/subunit&complex&that&acts&as&initiator&of&DNA&replication&o Requires&ATP&to&bind&ARS&elements&o Single/stranded&DNA&stimulates&ORC&to&hydrolyze&ATP&
• Replication&process:&o ORC&binds&to&an&origin&(to&ARC&elements)&o MCM&complex&binds&to&this&with&help&of&cdc6&o Initiator&complex&is&activated,&and&helicase&activity&opens&the&parental&strands&o SSB&binds&to&exposed&single&strands,&enlarging&bubble&o Pol&α/primase&synthesizes&first&RNA&primer&o Switch&to&elongation&by&DNA&polymerase&δ&o RNA&primer&is&synthesized&on&the&lagging&strand&o Two&replication&forks&
• Eukaryotes&contain&several&different&DNA&polymerases&o α,&δ,&ε&/>&Nuclear&DNA&o *&α&only&polymerase&that&associates&with&primase,&synthesizing&a&RNA/DNA&hybrid&o *δ&or&ε&used&for&processive&elongation&of&both&strands&o β&used&for&base/excision&repair&o Polymerase&switch/&Exchange&of&DNA&pol,&which&occurs&only&after&the&RNA/DNA&hybrid&is&
made&o γ&/>&Mitochondrial&DNA&
o DNA&polymerases&possess&two&unusual&features&! Syuntehsize&DNA&only&in&the&5’/>3’&direction&! Cannot&initiate&DNA&synthesis&
• This&presents&a&problem&solved&by&telomeres&• Eukaryotic&Lagging&Strand&synthesis&
o A&type&of&single&strand&DNA&binding&protein,&called&RPA,&binds&to&exposed&single&strand&of&the&future&lagging&strand&
o Replication&Factor&F&(RFC)&binds&to&the&primer&and&serves&as&a&clamp&loader&for&PCNA&sliding&clamp&
o pol&β&binds&to&the&PCNA&and&completes&the&Okazaki&fragments&to&a&final&length&• Okasaki&Fragment&processing&in&Eukaryotes&
o Primer&removal&carried&out&by&RNase&H&and&FEN1&! Rnase&H°rades&the&RNA&primer,&leaving&a&single&ribonucleotide&attached&! Flap&Endonuclease&1&(FEN1)&removes&this,&and&a&bit&more&(removing&possible&
mismatches)&! Gap&filled&by&pol&δ&extending&the&3’&end&of&the&Okazaki&fragment&
• Nucleosome&and&DNA&Replication&o Replication&doubles&the&amount&of&DNA&(histones&must&be&synthesized&before&
replication;&during&S&phase)&o Histones&assemble&into&octamer&structures&
• Telomeres&and&DNA&Replication&o Linear&eukaryotic&chromosomes&have&telomeres&at&both&ends&o Solves&the&DNA&polymerase&dilemma&o Rich&in&G&and&T&o Telomerase&
! A&ribonucleoprotein&complex&with&short&RNA&strand&as&an&integral&part&! Contains&protein&and&RNA&! Adds&new&6/nt&telomere&repeats&to&the&3’&end&of&a&DNA&chain&! Rinse&and&repeat&! YOUTUBE&
&
DNA*Repair*and*Recombination*
• DNA&damage&results&in&either&repair&or&tolerance&o Tolerance&
! Damage&sites&are&recognized&by&replication&machinery&before&they&can&be&repaired,&resulting&in&an&arrest&that&can&be&relieved&by&replicative&bypass&
o Repair&! Involves&excision&of&bases&and&DNA&synthesis.&Mispaired&bases&are&excised&as&
single&nucleotides&during&mismatch&repair&o The&cell&may&also&be&arrested,&or&lead&to&cell&death&
• Diseases&due&to&DNA&damage:&o Werner&Syndrome&
o Bloom&syndrome&o Ataxia&Telangiectesia&o Xeroderma&Pigmentosum&(XP)&o Cockayne&Syndrome&
• Fuckups:&o AP&site&
! Depurination&which&leaves&an&abasic&AP&site,&but&does¬&break&the&sugar/phosphate&backbone&
o O6/methyl&guanosine&! Methylation&at&O&! Has&a&different&pattern&of&H/bond&donor&and&acceptor&atoms&that&the&parent&
guanine&base,&resulting&in&pairing&with&T&instead&of&C&! Gives&rise&to&G/>A&! Fixed&by&AGT,&which&takes&the&methyl&group&from&the&O,&and&is&later°rade.&
Alternatively,&use&MGMT&(a&“suicide”&enzyme)&for&same&fix&o Crosslinking&of&adjacent&pyrimidines&
! Results&from&UV&light&! Ranked&as&Large&Lesions&
o Distortion&of&the&cross/linked&bases&! Made&by&a&cyclobutane&dimer&
• Base&excision&repair&(BER)&o Glycosylase&removes&base,&leaves&backbone&intact&o AP&endonuclease&cuts&backbone,&AP&lyase&remoes&sugar&o DNA&polymerase&fills&gap&o DNA&ligase&seals&nick&o Types&of&lesions&repaired&by&BER&
! Oxidative&lesions&! Deoxyuracil&! Various&alkylation&products&! *These&lesions&do¬&block&DNA&polymerases&! Spontaneous&depurination&
• Nucleotide&excision&repair&(NER)&o Double&excision&removes&damage&at&part&of&an&oligonucleotide&o DNA&polymerase&fills&gap&o DNA&ligase&seals&nick&o Repairs&large&lesions&
• DNA&glycosylases&o Hydrolyze&the&glycosidic&bond&of&their&corresponding&altered&base&to&yield&an&AP&site&o APE1/&Most&important&
! Recruits&DNA&pol&β&to&the&site.&This&pol&has&AP&lyase&activity&that&remoes&the&abasic&sugar,&leaving&a&single/nucleotide&gap.&With&its&DNA&polymerase&activity,&it&fills&this&gap.&
• Transcription/Coupled&Repair&o A&form&of&excision&repair&triggered&by&an&RNA&polymerase&complex&halted&at&a&lesion&
o Directs&the&immediate&repair&response&to&the&template&strand&of&transcribed®ions&• Direct&Repair&of&UV&damage&in&E.&coli&
o Photoreactivation&! Involves&enzyme&Photolyase&! Uses&energy&of&light&! Direct&reversal&of&damage&! Not&working&in&humans&
• Methyl/Directed&Mismatch&Repair&in&E.&coli&o Mismatch&Repair&(MMR)&
! MMR&deficiency&gives&rise&toµsatellite&instability&! MMR&occurs&close&to&the&replication&fork&
• Double&strand&repair&o Nonhomologous&end/joining&in&eukaryotes&
! Usedonly&in&emergencies&! Two&broken&ends&ofDNA&are&joined&together&! Creates&he&Holiday&Junciton&
• There&are&several&ways&to&bypass&parent&strand&lessions&(Review)&• DNA&repair&enzymes&
o A&lot&of&DNA&damage&/>&elevated&lvls&of&repair&enzymes&o Extreme&changes&in&cell’s&environment&/>&activates&genes&for&DNA&repair&(ex:&hs)&
• Cell&Cyle&and&DNA&repair&o Cell&cycle&is&delayed&if&there’s&DNA&damage&
! Ensures&that&DNA&damages&are&repaired&before&it÷s&• SOS&Response&
o Occurs&in&E.&coli&o Damage&to&DNA&triggers&SOS&response,&a&coordinated&change&in&gene&expression&that&
aids&in&recovery&from&damage&o Regulon/&A&group&of&operons&controlled&by&a&common&repressor&
• Recombination&o The&exchange&of&genetic&information&o Two&basic&types&
! Homologous&recombination&(aka&general&recombination)&• Reciprocal&• Occurs&between&paternal&and&maternal&chromosomes&
! Nonhomologous&recombination&• Site/specific&recombination&
o Seen&in&bacteria&o Integrates&a&plasmid&into&the&chromosome&
• Transposition&o Movement&of&specific&pieces&of&DNA&in&the&genome&o Simple&transposition&
! Sequence&is&excised&from&one&place&and&inserted&somewhere&else&
o Replicative&transposition&
! Copy&of&the&sequence&is&inserted&in&the&genome&without&losing&the&original©&
• Transposons&o A&transponsable&element&is&a&DNA&sequence&that&can&change&its&
position&within&the&genome&• In&Humans:&
o Lines&! Long&interspersed&elements&which&are&flanked&by&UTR&
and&target&side&duplications&o Sines&
! Short&interspersed&elements&that&represent&reverse/transcribed&RNA&molecules&originally&transcribed&by&RNA&pol&III&into&tRNA,&5S&rRNA,&and&other&snRNA&
o Retrotransposons&! Sequences&that&were&transcribed&into&RNA&and&then&
reverse/transcribed&and&integrated&back&into&a&random&site&in&the&genome,&using&a&virus/encoded&integrase&
! *“Retro”.&Think&of&Retro&virus.&o There&are&three&major&models&for&homologous&recombination,&all&having&a&common&
intermediate:&Holliday&junction&o Holliday&Model&
! Key&features:&• Homology&• Symmetry&of&both&breaks&and&strand&invasion&• Presence&of&a&four/stranded&“Holliday&Junction”&as&a&key&intermediate&
&
*
Cell*Cycle,*Apoptosis,*and*Cancer&
• Normal&Cell&Growth&o Cell&cycle:&Interphase&/>&Mitosis&
! G1/&Cell&growth,&cell&doubles&organelles;&longest&stage&! G0/&&Cells¬&actively&dividing;&Physiologically&active;&*Restriction&checkpoint&! S/&Genetic&material&replicates&! G2/&Protein&synthesis&for&mitosis;&RNA&synthesis;&Second&checkpoint&! Mitosis&(shortest&stage)&
• Prophase/&Two&DNA&molecules&(DNA&already&replicated);&Compacted&chromosomes;&Mitotic&spindles&forms;&Nucleoli&disappear&
• Prometaphase/&Astral&MT&appear;&chromosomes&moves&• Metaphase/&Metaphase&plate&(chromosomes&align&in&middle)&• Anaphase/&Sister&chromatids&separate&
o Anaphase&A/&Kinetochoreµtubule&shorten&
o Anaphase&B/&Polarµtubule&elongate&• Telophase/&Cleavage&furrow;&Nucleus&reforms;&Chromosomes&
decondencing;&Kinetochoreµtubules&disappear&• Cytokinesis/&Cleavage&of&cytoplasm&begins&on&Anaphase&and&completes&
before&Interphase;&*Contractile&ring÷s&cell&• *Mitosis&without&cytokinesis&leads&to&multinucleated&cells&• *Replication&separated&from&mitosis&leads&to&polytene&chromosomes&
! Meiosis/&Gametes;&Diploid&(2n)&/>&Haploid&(n)&• Meiosis&1&(four&phases:&Prophase&1&/>&Telophase&1)&
o Interphase&1/&Chromosomes&replicate&(S&phase);&Centriole&replicates&
o Prophase&1/&Homologous&recombination;&*Crossing&over&among&non/sister&chromatids&at&chiasmata;&Longest&phase&
o Metaphase&1/&Tetrads&align&at&plate;&Shortest&phase&o Anaphase&1/&Homologous&chromosomes&separate;&*Sister&
chromatids&are&still&attached&o Telophase&1/&*Each&pole&has&now&haploid&cells&
• Meiosis&2&=&Mitosis&(*Review*)&• Cell&cycle®ulation&
o Three&important&Checkpoints:&! G1&Checkpoint&(From&G0&to&G1)&
• Checks&for:&o Cell&size&o Nutrients&o Growth&factors&o DNA&damage&
• Cyclin&D&(G1)&• Cyclin&E&(from&G1&to&S)&
o *Aside:&Cyclin&A&(from&S&to&G2)&! G2&Checkpoint&(From&G2&to&M)&
• Checks&for:&o Cell&size&o DNA&damage&
• Cyclin&B&(From&G2&to&M)&! Spindle&Assembly&Checkpoint&(At&M)&
• Checks&for:&o Chromosome&attachment&to&spindle&
! CDk’s&(Cyclin&Dependent&Kinases)&• Regulates&cell&cycle&• Cyclins&are&constantly&synthesized&and°raded&• Cyclin&ubiquitination&by:&
o APC&(Anaphase&Promoting&Complex)&! Regulates&M/cyclin&
o SCF&! Regulates&G1/S&and&S/cyclin&complex&
! Regulation&of&E2F&• Rb&(Retinoblastoma)/&Tumor&suppressor&protein&
o Active/dephosphorylated/&Sequesters&TF&E2F&o Inactive/phosphorylated/&Releases&TF&E2F&for&transcription&of&S&
phase&genes&! p53&
• Activated&(phosphorylated)&by&DNA&damage&• ATM&is&activated&when&DNA&is&damaged&by&radiation.&This&activates&p53&
(a&TF)&• p53&is&activated&by&HAT&and&inactivated&by&Mdm2&(useful&for&cancer&
cells).&Mdm&2&is&inhibited&by&activated&Arf&(this&means&that&p53&will&be&activated)&
• High&p53&increases&/>&p21&(WAF)&activated&/>&arrest&of&cell&cycle&before&S&phase&to&repair&DNA&
o Alternatively,&it&induces&apoptosis&if&damage&is&irreparable&! Mitogens&
• GF&(Growth&Factors),&cytokines,&hormones&• MAPK&(Mitogen&activated&protein&kinases)&activates&transcription&
factors&• Induce&transcription&of&Cyclins,&Cdk’s,&E2F&TF&
• Apoptosis&o Programmed&cell&death&o Does¬&induce&inflammatory&or&an&Inmuno&response&(unlike&necrosis)&o Triggered&by&DNA&damage,&early&S/phase,&lack&of&GF,&and&death&signals&o Caspases&
! Degrade&proteins&and&promote&cell&death&! Sleeping&version:&Procaspase&(Zymogen)&! Activated&version:&Caspase&! Adaptor&proteins&bring&them&together&for&cleaving&each&other&! Caspases&contain&cysteine,&which&prefers&to&cut&at&aspartate&(Casp)&! Two&groups&of&caspases:&
• Initiator&caspases&(2,&8,&9,&10)&o Cleave&each&other&
• Effector&caspases&(3,&6,&7)&o Signal&lification&and&cellular&cleavage&
o Two&major&pathways&of&Apoptosis&! Death&Receptor&Pathway&(Extrinsic)&
• *Skip*&• Involves&death&receptors&that&results&in&procaspase&9&• Cascade:&
o FasL&binds&to&Fas&(DD&=&FADD);&or&TNF&binds&to&TNF/R1&(DD&=&TRADD)&
o Death&domains&activated.&Binding&of&Adaptor&proteins&that&recruit&caspases,&for&mutual&cleavage&
o FADD/TRADD&promotes&aggregates&of&procaspase&8&to&form&a&“Death&Inducing&Signaling&Complex”&(DISC)&
o Caspase&8&(initiator)&is&activated.&This&can&cross/over&to&the&other&pathway.&
! Receptor/regulated&(Intrinsic)&• *Mitochondrial&Pathway&• Regulated&by&BH&proteins&(Bcl/2&homology)&• Pro/apoptotic&BH&proteins&Bax&and&Bak&causes&cytochrome&C&to&leak&out&
of&mitochondria&• Cytochrome&C&+&Procaspase&9&+&Apaf1&=&Apoptosome&
o This&activates&the&caspase&cascade&• Bcl/2&and&Bcl/XL&promote&cell&survival&by&binding&to&Bax&and&Bak,&
preventing&membrane&permeabilization&• Bid&and&Bad&
o Promote&apoptosis&o Dissociate&Bcl/2&and&Bcl/XL&from&Bax&and&Bak&
• Smac/Diablo&o Comes&out&from&mitochondria&to&inhibit&XIAP&(apoptosis&
inhibitors)&o Activates&caspases&
• AIF&and&Endo&G&o Pass&from&mitochondria&to&nucleus&and°enerate&DNA&
o p53/induction&of&Apoptosis&! p53&increases&p21,&which&prevents&Cdk/promoted&cell&cycle&progression&! Excessive&p53&also&increases&concentration&of&pro/apoptotic&BH&proteins,&
resulting&in&apoptosis&• Cancer&
o Cell&Biology&of&Neoplastic&Transformation&! Neoplastic&Transformation/&Cells&that&proliferate&in&defiance&of&normal&growth&
controls.&Maybe&benign&or&malignant&! Malignant&type&infiltrate,&invade&and&destroy&surrounding&tissue&! Cancer&results&from&breakdown&of&signaling&mechanisms&that®ulate&normal&
cell&proliferation,&differentiation,&survival/apoptosis,&and&migration/invasion&o Molecular&Biology&of&Neoplasia&
! Proto/oncogene&• Normal&gene&that&can&undergo&a&genetic&change&to&become&cancerous&
! Oncogene&• Growth&inducer&• A&gene&that&causes&a&normal&gene&to&become&cancerous&• Mutant&overactive&form&of&a&proto/oncogene&
! Tumor&suppressor&gene&• Growth&inhibitor&• DNA&repair&protein&• A&recessive&mutation&in&an&inhibitory&gene,&thus&activating&gene&• Loss&of&function&causes&tumorigenesis&
o Cancer&Cells&! Divide&in&defiance&of&normal&constraints&! Invade&surrounding&tissues&(malignant&tumor&=&Adenocarcinoma)&! Damaged&DNA&! Increased&telomerase&activity&(Immortalization)&! Suppressed&differentiation&! Angiogenesis&! Aneuploidy&
o Cancer&Development&! Progression:&
• Hyperplasia/&An&increase&in&size&of&an&organ&due&to&increase&in&number&of&cells&
• Dysplasia/&An&abnormal&cellular&proliferation&in&which&there&is&a&loss&of&normal&architecture&and&orientation&
• Carcinoma&in&situ/&Malignant&neoplasm&of&epithelial&origin&that&has¬&penetrated&through&the&basal&lamina&
• Invasive&Carcinoma/&Tumor&cells&have&penetrated&the&basal&lamina&intothe&connective&tissue&underneath&but¬&spread&via&circulation&yet&
• Metastatic&Carcinoma/&Tumor&cells&have&spread&through&the&body&and&established&new&
! Tumor&Initiators&• Mutagens&that&cause&genetic&changes&• DMBA&(found&in&cigarette&smoke),&UV,&radiation&
! Tumor&promoters&• Not&mutagenic&• Stimulates&cell&proliferation&• Prolonged&exposure&cause&cancer&• Phorbol&esters&
o Cancer&is&a&result&of&dysregulated&gene&expression&! Mutation&in&the&promoter®ions&of&genes&
! Overexpression&or&deletion&of&genes&! Overexpression&of&transcription&factors&! Point&mutations,&insertions,&and&translocations&can&activate&or&inactivate&genes&! Epigenetics/&DNA&silencing&by&methylations&in&promoter®ions&can&silence&
tumor&suppressors&(which&is&bad)&o Cell&Cycle&Checkpoints&(Tumor&Suppressors)&
! Rb&protein&binds&and&inactivates&E2F&! p53&upregulates&21&and&pro/apoptotic&proteins&in&response&to&DNA&damage&! p21,&CKI,&cdkI&! BRCA&(Breast&Cancer&Susceptibility&gene),&involved&in&DNA&repair&! APC&(Adenomatous&Polyposis&Coli)&in&wnt&signaling&
• Considered&the&“gatekeeper”&gene&in&cancer&• FAP&is&caused&by&APC&mutations&• Inactivation&of&APC&gene&results&in&screw/ups&• APC&is&a&component&a&component°radation&complex&that°rades&
Beta&Catenin&for°radation&o If&Beta&Catenin&is&accumulated,&it&activates&TFs&of&different&
oncogenic&factors&o Good&Genes&gone&Bad&
! Oncogenic&Ras&• Has&activating&point&mutations&in&the>P&binding&domain.&This&renders&
the>Pase&domain&of&Ras&insensitive&to&inactivation&by&GAP&proteins&(therefore,&it’s&always&activated).&This&activates&MAPK&cascade,&which&in&turn&activates&TFs&that&induce&cell&cycle&progression&
! Oncogenic&Akt&• Inhibits&apoptosis&by&phosphorylating&and&ubiquitinating&pro/death&Bcl2&
family&member&BAD&o TGF/β&signaling&induces&cell&cycle&arrest&via&transcription&of&Ckis&(p21&included)&
&
Mendelian*Chp*6*
• Hemizygous&o Only&have&a&single©&of&each&gene&o Happens&in&males;&X/Y&o Dominance&and&recessiveness&does¬&matter&
• Hardy/Weinberg&o Large&population&o Random&mating&o No&mutations&o No&in&and&out&of&population&o No&selection&in&ability&to&survive&
o Only&two&alleles&(p&=&A&;&q&=&a)&o p&+&q&=&1&o (p&+&q)2&=&12&o p2&+&2pq&+&q2&=&1&
&• Bayesian&analysis&example&
o Picharle&con&un&guille&carbon&• Parental&diagnosis&
o Imprinted&disorders/&Modifications&(such&as&Methylation)&to&a&particular&gene&affects&its&expression.&This&may&lead&to&disorders&(???)&
Mendelian*Inheritance*Chp*7*
• Genetic&Heterogeneity.&o Clinical&heterogeneity/&“Mutaciones&en&un&gen&causan&diferentes&enfermedades”&o Allelic&heterogeneity/&“Diferentes&mutaciones&en&el&mismo&alelo&(gen)&causan&
enfermedad”&&o Locus&heterogeneity/&“Diferentes&gene&locus&causan&la&misma&enfermedad”&
• Phenocopy/&A&phenotype&that&resembles&a&genetic&disorder,&but&developed&in&the&absence&of&an&inherited&phenotype&
• Variations&in&dominant&phenotypes&o Penetrance&
! Likelihood&that&a&dominant&trait&will&manifest&itself&! Non/penetrance/&Failure&to&manifest.&Depends&on&sex,&age&and&modifier&genes.&
o Variable&expressivity&! Different&features&of&a&disorder&are&seen&in&different&members&of&the&same&
family&! “&Una&mutación&exactamente&igual&en&todo&el&mundo&se&puede&expresar&
diferente”&o Anticipation&
! A&type&of&variable&expressivity&associated&to&instability&repeat®ions.&With&successive&generations&the&repeats&lify,&resulting&in&more&severe&phenotypes.&(Tus&hijos&van&a&estar&mas&jodios&que&tu)&
o Huntington’s&disease&! Example&of&Anticipation&! CAG&is&repeated&
o Fragile&X&Syndrome&! Example&of&Anticipation&! CGG&on&the&X&chromosome&! *Most&common&form&of&inherited&mental&retardation&
o De&novo&mutations&! Mutations&may&occur&spontaneously&
! Larger&gene,&larger&chances&o Mosaicism&
! The&mutation&is&carried&in&a&proportion&of&cells&! May&be&somatic&(safe&to&procreate)&or&germline&(10%&risk&of&transmission)&
&o Imprinting&
! Silences&either&the&maternal&or&paternal&gene&via&methylation&• Alternative&method:&histone&modifications&
! UPD&(Uniparental&disomy)&• Beckwith/Wiedemann&syndrome&• Angelman&syndrome&
o Variations&in&recessive&phenotypes&! Frequently&occurring&genes&! Founders&populations&! Consanguinity&! Gene&expression&level&
o Mitochondrial&inheritance&! Affect&both&Males&and&Females&equally&! Mimics&X/linked&disorders&(transmitted&by&mothers),&but,&unlike&X/linked,&
affected&males&never&pass&it&onto&their&daughters&! *Mitochondrial&DNA&replication&is&more&error/prone&! Heteroplasmy/&Cellular&mosaicism;&mixed&population&of&normal&and&abnormal&
mitochondria&within&cells&! Mutation&results&in&respiratory&chain&defects&
o Modifier&genes&! Not&causative&of&disease&alone,&but&modify&the&severity&of&phenotypic&expression&
of&a&disease&
&
&
&
Chromosomal*Disorders&
• Karyotype&o Chromosome&analysis&arranged&in&pairs&o Banding&patterns&depends&on&stains&o Giesma&stain&is&most&common&o Dark&band&heterochromatic®ions&
! Genes¬&expressed&! AT&rich&
o Short&arm&=&p&(petit)&o Long&arm&=&q&o Human&chromosomes&are÷d&in&7&groups&+&sex&chromosomes&
• Molecular&Cytogens&o FISH&(Fluorescent&in&situ&hybridization)&
! Fluorescent&DNA&probe&specific&to&area&of&interest&o CGH&(Comparative&Genomic&Hybridiazation)&
! Reference&DNA&are&hybridized&simultaneously&to&metaphase&chromosomes&! Disadvantage/&Doesn’t&detect&balanced&rearrangements&
• Chromosomal&numerical&abnormalities&o Aneuploidy/&Abnormal&number&of&chromosomes&
! Trisomy&21,&18,&13&(Important&dates&in&life)&o Nondisjunction&
! May&happen&in&Meiosis&I&or&Meiosis&II&o Sex&chromosomal&aneuploidy&
! Turner&syndrome&• X/0&
! Klinefelter’s&syndrome&• XXY&• Guys&that&look&like&girls&
o Polyploidy&! Changes&in&the&number&of&complete&sets&of&chromosomes&! Usually&results&in&death&! Ex.:&Triploidy&(3n)&
• Chromosomal&structural&abnormalities&o Translocations&
! When&a&portion&of&a&chromosome&is&exchanged&with&another&non/homologous&chromosome&
! Can&be&either&Robertsonian&(a&fragment&that&fails&to&segregate&is&lost)&or&Reciprocal&
o Microdeletion/Microduplication&syndromes&! Deletions/&A&portion&of&the&chromosome&is&deleted&
• Williams&Syndrome&• DiGeorge&Syndrome&• Cri/du/chat&Syndrome&
! Duplication/&A&portion&of&the&chromosome&is&duplicated&o Inversions&
! A&portion&of&the&chromosome&has&broken&off,&turned&upside&down&and&reattached,&inverting&genetic&material&
o Rings&! A&portion&of&the&chromosome&has&broken&off&and&formed&a&circle&
o Isochromosome&! Formed&by&the&mirror&image©&of&a&chromosome&segment&including&the&
centromere&
*
*
RNA:*Transcription*and*RNA*Processing&
• Transcription&o Transcription&is&the&mechanism&by&which&a&template&strand&of&DNA&is&utilized&by&specific&
RNA&polymerases&to&generate&one&of&the&three&different&classifications&of&RNA.&o Three&main&RNA&classes:&
! mRNA&• Genetic&coding&templates&used&to&determine&amino&acid&incorporation&
order&• About&5%&of&total&RNA&• Spliced,&5’&cap,&polyA&tail&at&3’&(both&only&in&eukaryotes)&
! tRNA&• Form&covalent&attachment&to&individual&amino&acids&• Recognizes&sequences&of&mRNA&to&allow&correct&insertion&for&elongation&• About&15%&of&total&RNA&• Spliced,&trimmed,&modified,&and&CCA&sequence&added&at&3’&end&in&both&
prokaryotes&and&eukaryotes&! rRNA&
• Form&ribosomes&• Form&catalytic&domain&where&tRNA&enter&with&their&attached&amino&
acids&• About&80%&of&total&RNA&• Processed&by&endonuclease&cleavage&in&both&prokaryotes&and&
eukaryotes&• For&prokaryots,&there’s&a&single&RNA&polymerase,&with&multiple&sigma&factors&• For&eukarots,&there&are&3&RNA&polymerases&
o pol&I/&rRNA&(28S,&18S,&5.8S)&o pol&II/&mRNA&and&most&snRNA&o pol&III/&tRNA,&5s&rRNA,&and&some&snRNA&
• Transcription&Inhibitors&o Alpha/Amanitin&
! Produced&by&fungus&(death&cap&mushroom)&! Potent&inhibitor&of&RNA&pol&II&and&weak&inhibitor&of&RNA&pol&III&
o Actinomycin&D&and&Acridine&! Intercalate&between&successive&G=C&base&pairs&in&duplex&DNA&! Inhibit&transcriptional&elongation&in&prokaryotes&and&eukaryotes&
o Rifampicin&(aka&Rifampin)&
! Binds&to&the&β&subunit&of&bacterial&RNA&polymerase&! Blocks&promoter&clearance&(which&is&used&for&elongation)&! Used&for&tuberculosis&treatment&
o Dactinomycin&(Actinomycin&D)&! A&DNA&intercalating&agent&! Used&in&cancer&chemotherapy&
• Cockayne&Syndrome&o Caused&by&a&defect&in&the&ability&of&cells&to&repair&DNA&that&is&being&transcribed&o Three&types&of&Cockayne&Syndrome&(caused&by&mutations)&o Cockayne&Syndrome&B&(CBS)&protein&
! Subunit&of&eukaryotic&RNA&pol&II&! Involved&in&transcription/coupled&DNA&repair&! Enhances&rate&of&elongation&by&RNA&pol&II&
• Transcription&of&RNA&refers&to&reactions&catalyzed&by&a&DNA/dependent&RNA&polymerase&o All&RNA&polymerase&are&dependent&upon&a&DNA&template&in&order&to&synthesize&RNA&
• Gene&Structure&o Gene&
! Segment&of&DNA&that&functions&as&a&unit&to&generate&an&RNA&product&(a&polypeptide&chain)&
! Includes®ions&of&DNA&that®ulate&production&of&the&encoded&proton&(ex:&promoter,&which&is&found&just&before&the&start&point&for&transcription)&
o Order:&! Regulatory&sequences&/>&TATA&box&/>&Transcription&initiation&point&/>&Future&Cap&
site&/>&5’&UTR&/>&Translation&initiation&point&/>&Translation&termination&point&/>&3’&UTR&/>&AATAAA&(?)&box&/>&Future&Poly/A&site&/>&Transcription&termination&point&
o Promoter&! Region&that&contains&nucleotide&sequences&recognized&by&RNA&polymerase&! Start&site&of&transcription&! Determines&specificity&of&transcription&of&a&gene&! Order:&
• Eukaryotes&o 5’&/>&CAAT&Box&/>&Hogness&Box&(aka&TATA&Box)&(ATATAA)&/>&Start&
site&/>&3’&o Activators&bind&at&/35&Nucleotides&o Inhibitors&bind&at&TATA&box&
• Prokaryotes&o 5’&/>&/35&sequence&/>&Pribnow&Box&(TATAAT)&/>&Start&site&/>&3’&o /35&sequence&is&binding&site&for&activators&o Pribnow&Box&is&binding&site&for&inhibitors&
o Β/Thalassemia&! Type&of&anemia&due&to&mutations&in&the&TATA&Box&(A/>G&or&A/C)&! Also&due&to&point&mutation&that&changes&the&sequence&at&the&polyadenylation&
signal&site&
! Also&caused&by&homozygous&mutations&in&the&splice/junction&sequences&at&intron/exon&boundaries&
! Results&in&lower&than&normal&levels&of&β/globin&chains&• General&mechanism&of&RNA&synthesis&
o Elongation&by&addition&of&ribonucleotides&to&the&3’&–OH&end&! Mechanism&same&as&used&for&elongation&of&a&DNA&strand&! Release&of&PPi&
• General&properties&of&DNA/dependent&RNA&polymerases&o Polarity&
! RNA&polymerase&reads&DNA&template&in&3’/>5’&direction&while&synthesizing&RNA&in&the&5’/3’&direction&
o DNA&template&! Either&strand&of&DNA&double&helix&can&be&used&
o Fidelity&! RNA&polymerases&do¬&possess&3’/>5’&proofreading&
• Prokaryote&Transcription&o Prokaryotes&Sigma&Factors&
! Directly&contacts&the&promoter&sequence&! Determine&what&genes&are&transcribed&! Sigma&Factor&+&Core&Polymerase&=&Holoenzyme&
o Rho/dependent&termination&! Enzymatic&mechanism&in&prokaryotes&that&emnply&an&ATP&dependent&RNA/DNA&
helicase&to&unwind&and&separate&the&3’&end&of&the&transcript&from&the&DNA&template&
o Bacterial&Operons&produce&polycistronic&mRNAs&(mRNA&for&different&proteins&are&placed&one&next&to&the&other;&remember&lac)&
o Bacterial&rRNA&operons&contain&tRNA&genes&as&well&• Eukaryotic&Transcription&
o CTD&(Carboxy&Terminal&Domain)&! Before&transcription,&CTD&is&unphosphorylated&and&is&associated&with&the&
mediator&and&components&of&the&initiation&machinery&! Partial&phosphorylation&of&CTD&during&transcription&initiation&recruits&the&
capping&enzymes&to&cap&the&5’&end&of&the&transcript&! Further&phosphorylation&recruits&component&of&the&pre/mRNA&splicing&
machinery&! RNA&pol&II&reaches&termination&signals&and&factors&required&for&cleavage&and&3’&
polyadenylation&of&the&transcript&associate&with&the&CTD&and&recognize&sequences&in&the&RNA&
o Polyadenylation&requires&3’&end&cleavage&by&endonuclease&activity&and&poly/A&addition&by&polyadenylate&polymerase&
! PolyA&stablilizes&mRNA,&and&is&involved&in&translation&initiation&• Splicing&
o Spliceosome&
! A&ribonucleoprotein&complex&composed&of&multiple&snRNPs&that&facilitate&the&mRNA&intron&splicing&reaction&
! U1&subunit&is&particularly&important&• Lupus/&Caused&by&autoimmune&antibodies&against&U1&snRNP&
o There&are&many&alternate&ways&to&splice&the&hnRNA&(primary&transcript)&o The&rRNA&is&self/spliced&
• tRNA&Structure&(described&form&5’&to&3’)&o D&loop&on&the&far&left&side&o Anticodon&loop&further&down&o A&Variable&loop&a&little&bit&up/right&o Another&loop&on&the&far&right&side&o Amino&Acid&attachment&site&at&3’&end&(CCA)&
• Types&of&RNAs&transcribed&from&RNA&pol&III&include&o tRNA&o 5S&rRNA&o Severalµ&and&small&nucleolar&RNAs&
&
&
mRNA*Degradation*and*Non*Coding*RNAs&
• Elements&of&an&mRNA&that&affect&stability&o 5’/&cap&
! Protection&against&5’/exonuclease&o Stem&loop&
! Inhibition&of&translation&! Can&stabilize&mRNA&
o Coding®ion&determinant&! Can&mask&other&mRNA&elements&to&stabilize&untranslated&mRNA&
o Premature&termination&codon&! Target&transcript&for&nonsense/mediated&decay&(NMD)&
o Exon/exon&junction&! Binding&site&for&nuclear&shuttling&proteins/&determinant&for&NMD&
o AU/rich&elements&(ARE)&! Binding&site&for&destabilizing&or&stabilizing&proteins&
• Example&of®ulated&mRNA&turnover/Transferrin&receptor&mRNA&(TfR&mRNA)&o Remember&IRE&(Iron&Regulatory&Elements)&o IRE/BPs&without&iron&bind&to&IREs&on&TfR&mRNA&o This&stabilizes&the&mRNA&molecule&so&it&canstay&intact&for&longer&o In&low/iron&conditions,&IRE/BPs&allow&the&cell&to&keep&producing&transferrin&receptors&o In&high/iron&conditions,&this&does¬&happen,&so&mRNA&is°raded&
• Two&types&of&mRNA°radation&o mRNA&Decay&
! “Turnover”&
! Order:&• Poly&A&shortening&• Decapitation&• Exonucleotic&cleavage&
! Decay&factors&localized&to&P&bodies&o NMD&(Nonsense/mediated&Decay)&
! “Surveillance”&! Premature&stop&codons&can&target&a&transcript&for&rapid,&deadenuylation/
independent&decay&! If&stop&codon&is&far&upstream&from&the&last&Exon&Junction&Complex&(EJC,&
deposited&during&splicing),&the&stop&codon&is&labeled&as&premature&• NMD&is&triggered&
• RNA&interference&(RNAi)&o Mechanism&where&the&presence&of&certain&fragments&of&ds&RNA&interferes&with&the&
expression&of&a&particular&gene&which&shares&a&homologous&sequence&with&this&dsRNA&• Small&interfering&RNA&(siRNA)&
o A&short&double/strand&of&RNA&with&overhangs&o Transcribed&by&Pol&II&o Interfere&with&the&expression&of&genes&o Produced&as&part&of&the&RNAi&pathway&by&the&enzyme&Dicer&o Bind&to&a&ribonuclease&complex&called&RNA/induced&silencing&complex&(RISC)&
! Guides&the&small&dsRNAs&to&its&homologous&mRNA&target&o The&whole&complex°rades&mRNA&
! With&perfect&complementarity,&miRNA&can&cleave&the&target&! If¬&that&good,&then&it&just&represses&it&
• Long&non&coding&RNAs&(lncRNAs)&o Considered&as&non/protein&coding&transcripts&o Transcribed&by&Pol&II&o Can&target&different&aspects&of&gene&transcription&(activators/repressors,&RNA&pol&II,&etc.)&o Regulate&TF,&controlling&gene&expression&o ***lncRNA&far&exceed&number&of&protein&coding&mRNAs&o Can&be&categorized&according&to&their&proximity&to&protein&coding&genes&
! Sense&! Antisense&! Bidirectional&! Intronic&! Intergenic&
o Cis&Silencing&! Coat&gene&clusters&to&silence&their&expression&by&making&them&inaccessible&to&
transcription&machinery&o Trans&Silencing&
! Interact&with&chromatin&modified&proteins&to&epigenetically&silence&genes&at&another&locus&
&
&
Regulation*of*gene*expression*(Prokaryotes)&
• Introduction:&o Seven&processes&that&affect&the&steady&state&concentration&of&a&protein&
! Transcription&! Posttranscriptional&processing&! mRNA°radation&! Translation&! Posttranslational&processing&! Protein°radation&! Protein&targeting&and&transport&
o Principles&of&Gene&Regulation&! Housekeeping&genes&
• Genes&for&products&that&are&required&at&all×&• Expressed&at&constant&levels&
! Constitutive&gene&expression&• Constant&expression&
! Regulated&gene&expression&• When&cellular&levels&of&a&gene&product&rise&and&falls&in&response&to&
molecular&signals&o Inducible/&Increase&in&concentration&o Repressible/&Decrease&in&concentration&
• General&Mechanism&of&Regulation&of&Gene&Expression&in&Prokaryotes&o RNA&Polymerase&binds&to&DNA&at&promoters&
! Regulation&of&transcription&initiation&involves&changes&in&how&RNA&polymerase&interacts&with&a&promoter&
o Transcription&and&translation&occur&simultaneously&• Regulation&of&Gene&Expression&in&Prokaryotes&
o Units&of&transcription&in&Bacteria&! Operon/&Complete®ulatory&unit&of&a&set&of&clustered&genes.&Includes:&
• Structural&Genes&• Regulatory&Genes&• Control&Elements&(regulation&sites)&
• A&bacterial&operon&o Polycistronic&mRNAs&are&produced&o Cistron&=&ORF&(Open&Reading&Frame)&
! Starts&with&start&codon&(AUG)&and&ends&with&stop&codon&(ex.&UAA)&• The&Lac&Operon&
o Contains&3&adjacent&structural&genes&(LacZ,&LacY&and&LacA)&! Transcribed&in&unision&
o Promoter&is&called&lacP&
o Regulatory&gene&is&lacl,&which&codes&for&lac&repressor&! Not®ulated;&always&transcribed&at&low&rate&
o Lac&operator&(lacO)/&Binding&site&for&lac&repressor.&Lies&between&lacP&and&lacZ&o Regulation:&
! Presence&of&both&Lactose&and&Glucose&• Not&necessary&for&cel&to&metabolize&lac&for&energy&
! Absence&of&Glucose&and&presence&of&Lactose&• Need&to&make&use&of&available&lactose&for&energy&• In&absence&of&Glucose,&cAMP&is&made&• cAMP&binds&to&CAP&(cAMP&binding&protein)&• This&increases&affinity&of&CAP&for&promoter&• Binding&of&CAP&to&promoter&facilitates&binding&of&RNA&polymerase&
! *Catabolite&Repression&• The&metabolism&of&Glucose&prevents&the&activation&of&the&lac&operon&by&
causing&a&decrease&in&intracellular&cAMP&levels&• The&Tryptophan&Operon&
o Tryptophan&is&important.&If¬&present,&the&cell&needs&to&make&it.&o Contents&of&Operon:&
! A&promoter&(trpP)&! An&Operator&(trpO)&to&which&trpR&repressor&binds&! The&trp&Operon&is&always&turned&on&
• It&is&a&repressible&operon&o Regulation:&
! Tryptophan&acts&as&a&corepressor&• The&trp&repressor&does¬&bind&the&trp&operator&unless&it&is&complexed&
with&tryptophan&o *Complete&opposite&of&lac&operon,&where&the&lac&repressor&only&
binds&when&it&is¬&complexed&with&a&small&molecule&inducer&! trpR&is&shut&off&when&trpR&is&complexed&with&tryptophan&to&maintain&low&levels&
of&repressor&in&the&cell&o trp&Operon&Attenuation&
! Attenuation&acts&like&a&rho/independent&termination&site&• In&the&absence&of&tryptophan,&it&has&no&effect&on&transcription&
! Attenuator&providesRNA&polymerase&with&a&second&chance&to&stop&transcription&
&
Regulation*of*gene*expression*(Eukaryotes)*
• Transcription&Regulation&in&Eukaryotes&o Some&sequences&provide&sites&of&interactions&for&general&TFs&while&others&provide&
targets&for&specific&TFs&• Role&of&Chromatin&
o Access&to&eukaryotic&promoters&is&restricted&by&the&structure&of&chromatin&
o Transcriptionally&active&chromatin&has&an&increased&sensitivity&to&nuclease/mediated°radation,&including®ions&with&sequences&that&are&especially&sensitive&to&DNAseI,&called&hypersensitive&sites&(HS)&
• Properties&of&Active&Chromatin&o Deficient&in&H1,&the&linker&histone&o Its&core3&histones&tend&to&be&acetylated&or&ubiquinated&o Chromatin&remodeling/&Transcription&associated&structural&changes&in&chromatin&
• Histone&acetylation/&HATs&and&HDACs&o HAT&(Histone&Acetyltransferasem)&o HDAC&(Histone&Deacetylase)&
• Strength&of&histone&DNA&association&is&modified&by&acetylation&of&lysine&residues&in&the&N/terminus&of&histone&proteins&
o Acetylation&of&lysine&weakens&the&electrostatic&interactions&between&the&octamer&core&and&the&DNA&
o *Gene&activation&correlated&with&acetylation&o *Gene&silencing&associated&with&methylation&
• DNA&Methylation&o Occurs&in&CG&sequences&on&both&strands&(CG&islands)&o Methylation&of&DNA&correlates&with&lack&of&transcriptional&activity&
! Co/repressors&may&be&recruited&o Repressed&chromatin&is&hypermethylated&(heterochromatin)&o The&“histone&code”&
! A&series&of&modifications&on&histone&proteins&that&are&proposed&to®ulate&gene&activation.&Include&phosphorylation,&acetylation,&ubiquination,&and&methylation&
• Genomic&imprinting&o Imprinting&
! The&methylation&pattern&of&DNA&inherited&from&the&sperm&or&the&egg&that&correlates&with&choice&of&allelic&expression&
! Some&genes&are&expressed&only&from&the&maternal&genome&and&some&only&from&the&paternal.&Males&methylate&a&different&set&of&genes&than&females&
o Prade/Willi&and&Angelman&Syndromes&! Deletions&of&the&same®ion&are&found.&! If&deletion&is&inherited&from&father,&Prader/Willi&is&seen&! If&deletion&is&inherited&from&mother,&Angelman&is&seen&! Disease&occurs&when&a&gene&that&is&deleted&in&one©&is&methylated&in&the&
other&o Fragile&X&Syndrome&
! X/linked&! Due&to&trinucleotide&repeat&expansion&(CGG),&which&increase&hypermethylation&
of&the&promoter®ion&that&represses&transcription&of&FMR1&(this&makes&you&retarded)&
• Gene&Regulatory&Region&o Enhancers&
! Upsrtream&Activation&Sequences&
! Assist&initiation&of&transcription&! *Position&and&orientation&independent&! Transcription&Initiation&Complex&
• Made&by&activators&that&bind&to&enhancer&folds,&which&attracts&TFs&• Response&Elements&
o Promoter&elements&in&genes&responsive&to&common®ulation&o Examples:&
! Heat&Shock&Elements&(HSE)&! Glucocoritcoid&Response&Elements&(GRE)&! Metal&Response&Element&(MRE)&
o Found&in&the&promoter®ions&of&genes&whose&transcription&is&activated&in&response&to&these&circumstances&
• Structural&Motifs&in&DNA/Binding&Regulatory&Proteins&o Required&for&optimal&recognition&and&binding&to&nucleic&acids&o Include:&
! Helix/Turn/Helix&(two&α&helices&separated&by&a&sharp&β&turn)&! Helix/Loop/Helix&! Zn&Finger&! Leu&Zipper&(Leucine&Zipper;&one&side&is&hydrophobic,&and&the&other&is&charged)&
• *Review&Luciferase&YOUTUBE&video&
&
&
Molecular*Diagnostics&
• Restriction&Endonucleases&and&Restriction&Maps&o Restricton&endonucleases&
! Recgonginzes&short&palindromic&sequences&! Protects&bacteria&against&viruses&via&methylation&! Pueden&cortar&y&fragmentar,&creando&“Sticky&ends”&
o Restriction&Map/&May&be&constructed&when&specific&RE&sites&are&identified&in&DNA&• Recombinant&DNA&and&Cloning&
o DNA&from&different&sources&can&be&ligated&to&form&a&recombinated&DNA&• Recombinant&vectors&can&be&produced&in&significant&quantities&by&cloning&
o Cloning&vector/&A&DNA&carrier&(either&a&bacterial&plasmid&or&virus)&o Plasmid/&Mini/circular&DNA;&pBR322&was&the&first&plasmid&constructed&o Replicon/&Region&required&in&a&plasmid&for&stability;&contains&Origin&+&DNA®ulatory&
sequencers&• *Autogradiogram&???&(LOOK&FOR&YOUTUBE&VIDEO!)&• Probes&are&used&to&identify&DNA&sequences&• Gel&Electrophoresis&
o Larger&molecules&travel&slower&(stays&near&top)&o Smaller&molecules&travel&faster&(reach&further&down)&
• DNA&labeling&methods&o Used&to&identify&specific&sequencing&o Methods:&
! Nick&translation&! Random&Primer&Method&
• Denaturation&and&addition&of&base&pairs&(hybridization)&=&Probe&! Labeling&with&a&T4&Polynucleotide&Kinase&(a&PO&donor)&
• Southern&Blot&o Requires&denaturation&o Allows&for&the&detection&of&specific&DNA&sequences&within&a&complex&mixture&of&DNA&o Hybridization&with&probes&can&demonstrate&if&DNA&sequence&of&interest&is&present&in&the&
same&or&different®ions&of&the&genome&o Uses&gel&electrophoresis&for&detection&of&specific&DNA&sequence&in&a&sample&of&DNA&o Horizontal&Gel,&Capillary&transfer&
• Northern&Blot&o Used&for&detection&of&RNA&sequences;&technique&similar&to&Southern&Blot&o Horizontal&Gel,&Capillary&Transfer&
• Western&Blot&o Uses&gel&electrophoresis&for&proteins,&separating&them&based&on&size&and&shape&o Vertical&Gel,&Uses&Current&
• Microsatellites&o Aka&SSR&(Single&Sequence&Repeats)&
• DNA&Sequencing&Methods&o Ion&Torrent&Chenmistry&/>&Youtube&o Pyro&Sequencing&/>Youtube&
• Sanger&Dideoxy&Sequencing&Method&/>&Youtube&o Requires&labeling&on&the&5’&end&o Both&single&and&double&stranded&templates&can&be&sequenced&with&ease&
• Polymerase&Chain&Reaction&(PCR)&o Used&for&lification&o Uses&the&Taq&polymerase&o Requires:&
! DNA&nucleotides&! Template&DNA&(the&one&you&want&to&lify)&! Primers&(single&stranded&DNA)&! DNA&polymerase&(heat/stable)&
• Automated&DNA&Sequencing&/>&Youtube&o Combines&the&Sanger&DNA&Sequencing&and&PCR&in&the&Cycle&Sequencing&Method&
• Massively¶llel&sequencing&/>&Youtube&o Similar&to&Sanger,&but&with&fluorescently&labeled&terminators&o Reads&at&a&much&faster&rate&
o Drawback:&Sequence&read&are&much&shorter&• Pyrosequencing&/>&Youtube&
o Trademark:&Enzyme&Luciferase&(makes&light)&• Personal&Genome&Machine/&Ion&Torrent&(life&technologies)&Semiconductor&Sequencing&/>&
Youtube&o Fastest&sequencing&method&on&the&market&o H+&ions&are&released&through&base&incorporation.&Also&uses&pH&meter.&
• Ion&Proton&System&Higher&capacity&semiconductor&sequencer&/>&Youtube&• Ion&Torrent&/>&Youtube&• Use&of&specific&probes&for&mutation&detection&(dot&blot&hybridization)&
o One&probe&binds&to&the&mutant&allele,&and&the&other&to&the&normal&gene.&Both&are&lified&through&PCR&and&analyzed&
• Synthesis&of&cDNA&from&mRNA&(???)&• Real&Time&PCR&• Taqman&probes&are&“hydrolysis”&probes&
o Polymerization&o Displacement&o Hydrolysis&o Polymerization&Completed&
• Microarray&/>&Youtube&• Array&CGH&
o Used&to&detect&genomic©&number&variations&at&higher&resolution&o Used&to&create&virtual&karyotype&
• Affymetrix&Gene&Chips&/>&Youtube&o Oligonucleotides&are&synthesized&o Uses&process&of&photolithography&o Each&gene&is&represented&by&a&“probe&set”&
&
Protein*Synthesis&
&
Protein*Structure,*Function*and*Methods*of*Analysis*
&
Fibrous*Proteins*
• Extracellular&matrix&(ECM)&o Cells&are&embedded&in&it&and&its&composition&determines&the&tissue&characteristics&o Functions:&
! Cell&support,&tissue&shape&and&integrity&! Cell&migration&
! Protective&barrier&o ECM&Diversity&
! Connective&tissue&• Skin&• Tendon&and&cartilage&• Basal&lamina&
o Composition:&! Fibrous&structural&proteins:&Collagen,&Elastin,&Laminin&! Proteoglycans&(gel)&! Adhesion&proteins:&Fibronecdtin&
• Fibrous&Proteins:&o Collagen&
! Produced&primarily&by&fibroblasts,&muscle&cells&and&epithelial&cells&! Classified&based&on&structure&and&functional&properties&! Types:&
• Fibril/forming&o Skin,&Cartilage,&Muscle&
• Network/froming&o Basal&lamina&
• Others:&o Non/fibrillar&collagens,&etc.&
! Collagen&composition&and&structure&• Insoluble&glycoprotein&• Triple&helix:&3&polypeptide&(α)&chains&• Gly/X/Y&
o X&=&Arginine&or&Proline&o Y&=&Proline&
• Left&hand&helix,&forms&fibers&(rope/like&structure)&! Disorders&of&Collagen&
• *Osteogenesis&Imperfecta&(OI)&o Brittle/bone&disease&o Most&due&to&de&novo&mutations&o OI&type&1&introduce&premature&termination&codons.&Collagen&is&
made&but&the&amount&of&the&trimer&manufactured&is&diminished&o OI&type&2/4&has&mutations&that&substitute&the&glycine&&
• Ehlers/Danlos&syndrome&(EDS)&o Characterized&by&skin&hyperextensibility/fragility&o Nonsense/mediated&decay&of&the&mutant&mRNA&
! Diminished&amounts&of&Collagen&V&• Alport&Syndrome&(AS)&
o Glomerulonephritis&o Mutation&in&Collagen&IV&
• Atopic&Dermatitis&(AD)&o Chronic&inflammatory&skin;&allergic&disease&
• Non/genetic&disorders&of&Collagen&o Scurvy&
! Vitamin&C&deficiency&! Proline&/>&Hydroxyproline&! Weak&collagen&! Leads&to&Cancer&
! Type&1&Collagen&• Most&abundant&protein&in&mammals&• Major&component&of&connective&tissue&• Contains&two&α1&chains&and&a&single&α2&chain.&
o *If&the&number&of&available&pro&α1&chains&decreases,&the&amount&of&the&trimer&formed&is&diminished&because&no&more&than&one&pro&α2&chain&can&be&accommodated&per&molecule&
o OI&is&caused&by&a&mutation&in&Type&1&collagen,&leading&to&either&reduced&synthesis&or&abnormal&collagen&formation&
o Elastin&! Has&fibillin&1&and&2&at&its&core&! Provides&structural&integrity&and&function&to&tissues&requiring&reversible&
extensibility&or&deformability&• Lung,&Skin,&Blood&vessels&
! Stretching&is&mediated&by&water&exposure&to&hydrophobic&residues&and&the&associated&entropy&
! Synthesis&and&Composition&• Precursor:&Tropoelastin&
o Composed&of&Lys,&Ala,&and&hydrophobics&! Elastin&Defects&
• Williams&Syndrome&o Cardiovascular&disease&o Elastin&gene&deletion&
! Elastin&insufficiency&leads&to&smooth&muscle&hypertrophy&and&arterial&stenosis&
! Fibrillin&Defects&• Marfan&Syndrome&
o Mutation&in&the&fibrillin&1&gene&o Autosomal&dominant&inheritance&o Issues&with&eye,&skeletal&and&cardiovascular&
o Laminin&! Abundant&basal&membrane&protein&! Heterotrimeric&protein&composed&of&alpha,&beta&and&gamma/subunits&! Associates&to&Type&IV&Collagen&! Synthsized&at&ER,&then&chain&association&within&the&Golgy,&secreted&out,&and&
extracellular&self/association/&disulfide&linkage&formation&for&stability&! Disorders&
• Junctional&Epidermolysis&Bullosa&
• Congenital&muscular&dystrophy&• Proteoglycans&(Mucopolysaccharides)&
o Up&to&95%&carbohydrate&o Polysaccharides&of&glycosaminoglycans&(repeated&disaccharide&units)&attached&
covalently&to&a&core&protein&o Degraded&in&lysosomes&o Functions:&
! Flexible&support&that&allows&diffusion&of&ions,&proteins&and&cells&! Interacts&with&ECM&proteins:&collagen,&elastin,&fibronectin,&and&laminin&! Found&in&interstitial&connective&tissues:&synovial&fluid,&cartilage&
o Synthesis&! Synthesized&in&ER&and&continued&in&Golgi&! Sulfation&occurs&! Secretion&from&cell&! Large&aggregates&form&! Binds&to&fibronectin&(which&is&attached&to&integrin)&and&collagen&
o Proteoglycan&Degradation&! Gycoproteins&including&proteoglycans&and&glycolipids&and&are&endocytosed&and&
degraded&within&lysosomes&• Lysosomal&proteases&digest&the&protein&component&• Lysosomal&glycosidases°rade&the&carbohydrate&component&
! Disorders&of&proteoglycans°radation:&• Mucopolysaccharidoses&
o Deficiencies&in&lysosomal&glycosidases&o Accumulation&of&carbohydrates&within&the&lysosomes&
• Integrins&o Principal&membrane&receptors&that&link&ECM&to&the&internal&cytoskeleton&o Functions&as&cellular&anchoring&structures&as&well&as&in&cell&signaling&o Composed&of&α&and&β&subunits&o Integrin&defects&
! Leukocyte&adhesion&deficiency&(LAD)&• Leukocytres&are&unable&to&activate&integrins&for&their&adhesion&to&
vascular&endothelial&cells&at&sites&of&infection&• Caused&by&mutations&in&the&β2/integrin&• Integrin&blocking&drugs&are&being&designed&to&treat&autoinmmune&
disorders&and&inflammation&• Adhesion&Proteins&
o Extracellular&glycoproteins&that&simultaneously&bind&to&collagen,&proteoglycans&and&integrins&
o Fibronectin&is&major&adhesion&protein&! Identified&as&defective&in&tumor&cells,&allowing&for&abnormal&movement&within&
the&ECM&and&metastasis&• Matrix&Metalloproteinases&
o Zinc&containing&proteases&that&cleave&all&proteins&of&the&ECM&
o Present&in&an&inactive&form&containing&a&propeptide&(Cys&+&Zinc&on&active&site)&o Important&for&cell&migration&and&tissue&remodeling&during&growth&and&differentiation&o Regulated&by&TIMPs&(Tissue&Inhibitors&of&Metalloproteinases)&o Clinical&Case:&Sis&Lupus&
! Underlying&autoinmmune&disorder&causing&cartilage°radation&
&
Emphysema&
• Emphysema&o Chronic&infection&of&the&bronchial&tubes&o Stretching&and&destruction&of&walls&(loss&of&elasticity)&
• Chronic&Obstructive&Pulmonary&Disease&(COPD)&o Cigarette&smoking&accounts&for&80/90%&of&cases&o Diagnosed&with&a&Spirometer&o A&/>&G&on&the&MMP12&promoter&o FEV1&is&important&
• Nutritional&Factors&in&COPD&o Fibers&good.&Meat&bad.&
• Linking&Genetics&and&Environmental&Risk&Factors&o COPD&and&asthma&differ&in&pattern&of&inflammation&and&inciting&agents&
! Asthma/&Allergens&! COPD/&Cigarette&smoke&
• MMP&o Proteases&that°rade&extracellular&matrix&molecules&
• Alpha/&1&Antitrypsin&(AAT)&o Protein&made&in&liver&o Released&to&bloodstream&o Deficiency&causes&illness&
! Lung&disease&(COPD)&o 120&different&variations,&or&alleles,&of&the&serpina1&gene&(which&codes&for&AAT)&
! M&is&most&common&! Z&contains&mutation&
• Glu&/>&Lys&• Causes&deficiency&• Leads&to&accumulation&of&proteins,&which&damages&liver&• Up&to&¼&in&PR&may&carry&one&Z&allele&• Qualified&for&lung&transplant&
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