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*

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*
o

&
*
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&(Chargaffs&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&

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&
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)&
! &synthesizes&DNA&
! &is&clamp&
! &is&clamp/loader&
DNA&pol&II,&IV&and&V/&DNA&repair&
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)&

o
o

Catenanes&are&separated&by&topoisomerases&
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&
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&
Bacterial&DNA&replication&is&coordinated&with&cell&division&
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&/primase&synthesizes&first&RNA&primer&
o Switch&to&elongation&by&DNA&polymerase&&
o RNA&primer&is&synthesized&on&the&lagging&strand&
o Two&replication&forks&
Eukaryotes&contain&several&different&DNA&polymerases&
o ,&,&&/>&Nuclear&DNA&
o *&&only&polymerase&that&associates&with&primase,&synthesizing&a&RNA/DNA&hybrid&
o *&or&&used&for&processive&elongation&of&both&strands&
o &used&for&base/excision&repair&
o Polymerase&switch/&Exchange&of&DNA&pol,&which&occurs&only&after&the&RNA/DNA&hybrid&is&
made&
o &/>&Mitochondrial&DNA&

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&&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&&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&
o

&
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&&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&cells&environment&/>&activates&genes&for&DNA&repair&(ex:&hs)&
Cell&Cyle&and&DNA&repair&
o Cell&cycle&is&delayed&if&theres&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.&
There&are&three&major&models&for&homologous&recombination,&all&having&a&common&
intermediate:&Holliday&junction&
Holliday&Model&
! Key&features:&
Homology&
Symmetry&of&both&breaks&and&strand&invasion&
Presence&of&a&four/stranded&Holliday&Junction&as&a&key&intermediate&

o
o

&
*
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&
! CDks&(Cyclin&Dependent&Kinases)&
Regulates&cell&cycle&
Cyclins&are&constantly&synthesized&and&degraded&
Cyclin&ubiquitination&by:&

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,&Cdks,&E2F&TF&
o

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&
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&
Cancer&Cells&
! Divide&in&defiance&of&normal&constraints&
! Invade&surrounding&tissues&(malignant&tumor&=&Adenocarcinoma)&
! Damaged&DNA&
! Increased&telomerase&activity&(Immortalization)&
! Suppressed&differentiation&
! Angiogenesis&
! Aneuploidy&
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&
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)&
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&
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,&its&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&
TGF/&signaling&induces&cell&cycle&arrest&via&transcription&of&Ckis&(p21&included)&
!
!
!
!

o
&

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&

Only&two&alleles&(p&=&A&;&q&=&a)&
p&+&q&=&1&
(p&+&q)2&=&12&
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&(???)&
o
o
o
o

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&mutacin&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 Huntingtons&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&
Mosaicism&
! The&mutation&is&carried&in&a&proportion&of&cells&
! May&be&somatic&(safe&to&procreate)&or&germline&(10%&risk&of&transmission)&
&
Imprinting&
! Silences&either&the&maternal&or&paternal&gene&via&methylation&
Alternative&method:&histone&modifications&
! UPD&(Uniparental&disomy)&
Beckwith/Wiedemann&syndrome&
Angelman&syndrome&
Variations&in&recessive&phenotypes&
! Frequently&occurring&genes&
! Founders&populations&
! Consanguinity&
! Gene&expression&level&
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&
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/&Doesnt&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&
! Klinefelters&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&

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,&theres&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&&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 /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&/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&

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&
o

&
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&

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&
o

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&&helices&separated&by&a&sharp&&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&()&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&1&chains&and&a&single&2&chain.&
o *If&the&number&of&available&pro&1&chains&decreases,&the&
amount&of&the&trimer&formed&is&diminished&because&no&more&
than&one&pro&2&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&

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&
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&&and&&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&2/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
o
o
o

Present&in&an&inactive&form&containing&a&propeptide&(Cys&+&Zinc&on&active&site)&
Important&for&cell&migration&and&tissue&remodeling&during&growth&and&differentiation&
Regulated&by&TIMPs&(Tissue&Inhibitors&of&Metalloproteinases)&
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&&in&PR&may&carry&one&Z&allele&
Qualified&for&lung&transplant&