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&divided&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&regulation&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&region&

• HU&binds,&causing&the&region&to&wrap&around&the&DnaA&protein&and&separates&the&AT/rich&region&

! 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&degrades&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&not&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&degrade.&

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&not&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&regions&• 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&to&microsatellite&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&divides&• 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&copy&

• 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&not&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/&Kinetochore&microtubule&shorten&

o Anaphase&B/&Polar&microtubule&elongate&• Telophase/&Cleavage&furrow;&Nucleus&reforms;&Chromosomes&

decondencing;&Kinetochore&microtubules&disappear&• Cytokinesis/&Cleavage&of&cytoplasm&begins&on&Anaphase&and&completes&

before&Interphase;&*Contractile&ring&divides&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&regulation&

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&degraded&• 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&not&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&amplification&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&degenerate&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&regulate&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&not&penetrated&through&the&basal&lamina&

• Invasive&Carcinoma/&Tumor&cells&have&penetrated&the&basal&lamina&intothe&connective&tissue&underneath&but&not&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&regions&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&regions&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&degradation&complex&that&degrades&

Beta&Catenin&for&degradation&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&GTP&binding&domain.&This&renders&

the&GTPase&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&copy&of&each&gene&o Happens&in&males;&X/Y&o Dominance&and&recessiveness&does&not&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&regions.&With&successive&generations&the&repeats&amplify,&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&regions&

! Genes&not&expressed&! AT&rich&

o Short&arm&=&p&(petit)&o Long&arm&=&q&o Human&chromosomes&are&divided&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&copy&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&regions&of&DNA&that&regulate&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&not&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 Several&micro&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&region&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&regulated&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&not&happen,&so&mRNA&is&degraded&

• Two&types&of&mRNA&degradation&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&degrades&mRNA&

! With&perfect&complementarity,&miRNA&can&cleave&the&target&! If&not&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&degradation&! Translation&! Posttranslational&processing&! Protein&degradation&! Protein&targeting&and&transport&

o Principles&of&Gene&Regulation&! Housekeeping&genes&

• Genes&for&products&that&are&required&at&all&times&• 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&regulatory&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&regulated;&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&not&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&not&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&not&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&degradation,&including&regions&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&regulate&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&region&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&copy&is&methylated&in&the&

other&o Fragile&X&Syndrome&

! X/linked&! Due&to&trinucleotide&repeat&expansion&(CGG),&which&increase&hypermethylation&

of&the&promoter&region&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&regulation&o Examples:&

! Heat&Shock&Elements&(HSE)&! Glucocoritcoid&Response&Elements&(GRE)&! Metal&Response&Element&(MRE)&

o Found&in&the&promoter&regions&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&regulatory&

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&regions&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&amplification&o Uses&the&Taq&polymerase&o Requires:&

! DNA&nucleotides&! Template&DNA&(the&one&you&want&to&amplify)&! 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&parallel&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&amplified&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&copy&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&degrade&the&carbohydrate&component&

! Disorders&of&proteoglycans&degradation:&• 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&degradation&

&

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&degrade&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&