MCB250Exam3Notes - MCB 250 exam 3 notes PDF

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MCB 250 exam 3 notes ...

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Lecture'20:'Homologous'Recombination;'Non-homologous'End'Joining' Repairing*double*stranded*DNA*lesions*or*breaks* o Involves*retrieving*sequence*information*from*undamaged*DNA* § Damaged*DNA*goes*through*recombination* Homologous*recombination* o DNA*is*cut*and*rejoined*à*recombination* o Takes*place*in*all*organisms* o Important*for* § DNA*repair*–*double*strand*breaks* § Meiosis*in*eukaryotes** • Crossovers*between*homologous*chromosomes*required*for* proper*chromosome*segregation*and*for*generating*diversity*in* gametes* § Transmission*of*genes*between*bacterial*strains* ICLICKER* o Photoreactivation'is*a*mechanism*of*DNA*repair*that*does*NOT*involve*breaking* of*a*phosphodiester*bond* Double*strand*DNA*Breaks*Initiate*Recombination* o DSBs*in*DNA*can*be*caused*by* § Oxidative*damage* § Radiation*damage* § Cellular*nucleases* Holliday*Junctions* o Generation*of*Two*Holliday*Junctions* § Requires*DNA*synthesis* § A*branched*nucleic*acid*structure*that*contains*four*double*stranded* arms*joined*together* • Key*intermediate*in*many*types*of*genetic*recombination,*as*well* as*double*strand*repair* o Branch*Migration*of*a*Holiday*Junction*–*Second*Step* § 3’*end*invades*the*homologous*duplex* § Process* • Base*pairs*on*homologous*DNA*strands*are*consecutively* exchanged*at*the*Holliday*junction,*moving*the*branching*point* up*or*down*the*DNA*sequence* o Resolution*of*a*Holliday*Junction* § Many*proteins*are*able*to*recognize*and*distort*the*Holliday*junction* • HJ*can*occur*in*two*ways* Enzymes*Responsible*for*Homologous*Recombination*in*E.*coli* o The*recombination*process*is*catalyzed*by*proteins* o More*than*one*pathway* o Primary*pathway* § RecA*–*required*for*all*pathways* • Forms*filaments*of*ssDNA*

Mediates*strand*invasion* Mutants*DON’T*do*homologous*recombination* Conserved*through*evolution*–*the*eukaryotic*version*is*Rad51* Initially*bids*to*a*single*strand*and*forms*a*filament*5’*to*3’* Searches*for*homology*and*initiates*strand*invasion*and*duplex* formation* • Requires*at*least*50*bp*of*homology* RecBCD* o Generates*a*3’*overhang*to*allow*strand*invasion* o Helicase/nuclease*that*binds*to*double*strand*break* o Helicase*unwinds*and*nuclease*degrades*both*strands* • RecBCD*encounters*chi*site* o Dramatically*overrepresented*in*the*e*coli*genome* • Stops*degrading*the*strand*with*3’*end,*and*becomes*more*active* degrading*the*strand*with*the*5’*end* • Generate*the*3’*tail*needed*to*initiate*recombination* RuvABC* • • • • •

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Lecture'21:'What'is'a'gene?' -

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The*Central*Dogma* o A*gene*is*a*region*of*DNA*that*contains*information*for*the*synthesis*of*a* particular*RNA*and/or*protein* § DNA*is*an*information*storage*molecule* § It*is*the*gene*products*(RNA*and*proteins)*that*carry*out*other*essential* functions*within*the*cell* What*do*geneticists*do?* o Discover*new*genes* § The*existence*of*genes*was*first*shown*by*the*experimental* demonstration*that*same*naturally*occurring*variations*in*the*structure* or*color*of*living*organisms*could*be*inherited*from*one*generation*to*the* next* o Determine*gene*functions* § In*addition*to*identifying*previously*undescribed*genes,*mutant*screens* gave*insight*into*the*gene’s*function* • Loss-of-function*mutations*of*a*gene*à*cause*of*mutations* o Characterize*the*molecular*structure*of*genes*and*gene*products* Gregor*Mendel* o Showed*that*heritable*traits*are*controlled*by*genes*that*are*passed*from*one* generation*to*another* o Designed*experiments*in*the*pea*plant* § Observed*that*individuals*can*be*pure-breeding*(heritable*by*definition)* for*traits* o Monohybrid*Cross*

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Cross*between*two*true*breeding*parents* Way*to*determine*whether*the*organism*that*expressed*a*dominant*trait* is*heterozygote*or*homozygote* o Mendel’s*rules*of*inheritance* § Inheritance*of*genes*if*under*the*control*of*genes*that*occur*in*pairs* • Because*they*are*diploid* o The*same*genes*are*present*in*2*copies*located*on*pairs*of* homologous'chromosomes* § When*two*different*alleles*were*paired,*one*member*of*the*pair*was* dominant*to*the*other* § Individuals*could*be*homozygous*or*heterozygous*for*a*dominant*allele* • They*would*express*the*same*dominant*phenotype* • Genes*are*segregated*during*gamete*formation*and*sort*randomly* in*the*next*generation* o Gene*vs*Allele* § Breast*cancer*gene* • BRCA1*&*BRCA2* o Everyone*has*them*–*some*have*the*allele*that’s* associated*with*a*higher*risk*of*breast*and*ovarian*cancer* § Discovery*of*dominant*and*recessive*alleles*had*an*important*implication* • Two*individuals*with*the*same*phenotype*could*have*different* genotypes* Meiosis* o During*meiosis,*the*cell*segregates*homologous*chromosomes*from*one*another* How*is*DNA*inherited*when*cells*divide?* o Mitosis* § Accounts*for*nearly*all*of*the*cell*divisions*in*your*body* § Chromosomal*DNA*is*faithfully*replicated*and*identical*copies*of*the* genome*are*passed*along*to*both*daughter*cells* o Meiosis* § Chromosomal*DNA*is*subdivided*unequally*between*daughter*cells,*with* some*cells*inheriting*maternal*alleles*and*other*paternal*alleles* § Germs*cells:*gametes*produced*in*animals*and*plants* Punnett*Square* o Monohybrid*cross* o Dihybrid*cross* § Involves*2*genes* § 9:3:3:1*ratio*in*F2* § different*genes*did*appear*to*be*inherited*independently** • two*reasons*why* o non-homologous*chromosomes*are*sorted*independently* at*meiosis**

o homologous*chromosomes*undergo*genetic* recombination*(crossing*over)*during*meiosis* -

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Independent*Assortment* Homologous*recombination* o Produces*recombinant*chromosomes*containing*a*mixture*of*maternal*and* paternal*alleles* Genetic*screen* o Method*for*discovering*new*genes* Mutagenesis* o An*organism*was*exposed*to*radiation*or*chemical*mutagens* o Essentially*random*process* § Every*chromosome*in*every*cell*experiences*a*unique*set*of*mutations* Mutant*phenotypes* o Abnormal*features*resulting*from*the*induced*mutations*

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Lecture'22:'Molecular'Analysis'of'Gene' Sanger*Sequencing* o Dideoxy*chain*termination* • All*DNA*polymerases*require*an*annealed*primer*and*a*template* strand* • As*the*primer*grows,*the*template*is*copied* • DNA*synthesis*is*in*the*5’*to*3’*direction* o 3’*end*is*extended*(grows)* § DNA*can*be*synthesized*in#vitro*(outside*the*cell)*by*annealing*a* complementary*primer*to*multiple*copies*of*a*ssDNA*template*strand* and*then*using*the*DNA*polymerase*core*enzymes*to*elongate*the*primer* § Chain*termination*sequencing*relies*upon*chemically*modified* nucleotides*which*contain*the*sugar*dideoxyribose* • ddNTPs*lack*the*3’-OH*group* o cannot*form*phosphodiester*bond*with*downstream* nucleotides* § chain*termination*sequencing* • small*%*of*nucleotides*added*to*the*DNA*synthesis*reaction*are* ddNTPs* § To*sequence*a*DNA*template* • One*performs*4*parallel*DNA*synthesis*reactions* o Each*contains*1*of*4*nucleotides* § Separated*in*gel*electrophoresis* § Requires'billions'of'copies'of'the'same,'relatively'pure'nucleotide' sequence' • DNA*Cloning:*that*made*it*possible*to*amplify*a*single*DNA* molecule*into*a*limitless*number*of*identical*copies' o Use*bacterial*plasmids*to*clone'

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Autonomously*replicating*extrachromosomal*DNA' Plasmid*is*replicated*and*its*genes*expressed*in* parallel*with*the*bacterial*chromosome' § Easily*purified' • Plasmid*is*much*smaller*than*bacterial* chromosome' o Two*can*be*readily*separated*once* cell*is*lysed' o Two*step*procedure' § DNA*sequence*of*interest*is*covalently*ligated*into* a*vector,*thus*creating*a*molecule*of*recombinant* DNA' § Recombinant*DNA*vector*is*transformed*into*a* compatible*host*cell' Cloning*Vector:*plasmid*pBR322' o Features*of*all*cloning*sites' § An*origin*of*replication*recognized*by*the*host*cell' § One*or*more*selectable*marker*gene(s)' § Cloning*sites*that*can*be*used*to*insert*a*DNA*sequence*of*interest' Once*a*single*molecule*of*unknown*DNA*has*been*ligated*into*a*plasmid*vector,*it*can* be*readily*sequenced' o The*recombinant*plasmid*is*transformed*into*an*E*coli*cell' o The*transformed*E*coli*is*grown*on*nutrients*to*produce*billions*of*identical* copies*of*that*plasmid' o Plasmid*DNA*is*purified*from*bacteria,*and*the*insert*sequenced*using*a*flanking* DNA*primer' Multiple*Cloning*Sites*(MCS):*large*number*of*restriction*enzymes*sites*grouped* together' o DNA*Libraries' § Mixture*of*many*different*DNA*sequences*that*were*cloned* simultaneously*into*a*large*number*of*host*cells' • Genomic*DNA*library:*contains*all*of*the*DNA*sequences*present* within*the*chromosomes*of*the*organism' o Constructing*a*library*from*genomic*DNA' § Genomic*DNA*digested*by*restriction*enzyme*into*millions*of*discrete* fragments' § Millions*of*copies*of*plasmid,*digested*with*a*compatible*restriction* enzyme' § Each*transformed*bacterium*grows*into*a*single*colony*on*the*plate' • All*colonies*contain*the*same*plasmid,*but*each*one*carries*a* different*DNA*insert' Analytic*bioinformatics' o Assemble*all*the*millions*of*sequences*in*order' o Scan*through*and*identify*stretches*of*“open*reading*frames”'

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o Look*for*start*and*termination*sequences*to*figure*out*which*open*reading* frames*belong*to*the*same*gene' o Compare*to*known*sequences' Forward*and*Reverse*Genetics' o Forward*genetics' § Discover*mutant*phenotypes' § Find*the*gene' § Sequence*DNA' o Reverse*genetics' § Sequence*entire*genome' § Find*the*gene' § Mutate*gene*and*learn*phenotype' PCR' o Amplify*specific*DNA*sequences*without'having*to*clone*DNA*into*cells' o Fast*à*can*be*performed*in*one*or*few*hours' o Can*amplify*a*single*DNA*sequence*from*minute*or*impure*samples' o One*cycle' § A*dsDNA*template*is*denatured*by*elevated*temperature*in*the*presence* of*DNA*pol;*dNTPs,*2*DNA*primers*complementary*to*opposite*strands* (94*degrees*à*denatures*the*double*stranded*DNA)' § Solution*is*cooled.*As*it*cools*the*primers*anneal*to*the*complementary* template*sequences*(40-60*degrees)' § At*its*optimal*temperature*DNA*polymerase*elongates*each*primer,* synthesizing*a*sequence*complementary*to*the*associated*template* strand*(72*degrees)'

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Lecture'23:'Molecular'Analysis'of'Gene'Expression* Synthesis*of*first*strand*of*cDNA*from*a*eukaryotic*mRNA* o Steps* § Purify*RNA*from*tissue* § Anneal*oligo-dt*DNA*primers* • Preferentially*anneal*to*polyA*tail*of*mRNAs* § Use*reverse'transcription*to*elongate*DNA*primers* • DNA*pol*that*uses*RNA*as*template* o Requires*primer,*template*junction*to*initiate*synthesis* § Degrade*RNA*strand*with*RNase* o First*strand*cDNA*–*used*as*template*to*produce*double*stranded*cDNA*(DNA* molecule*complementary*to*RNA)* § Double*stranded*cDNA*–*can*be*cloned*into*a*plasmid* o Generates*cDNA*library* § Used*to*screen*for*particular*genes*via*DNA*hybridization* • Needs*to*contain*complementary*bp*sequences* Genomic*vs*cDNA*library* o Genomic*

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Cleaved*in*to*random*fragments* • No*fixed*relationship*between*clones*and*gens* Contains*all*the*DNA*in*a*genome* • Including*sequences*that*aren’t*transcribed*or*translated*

o cDNA* § each*clone*derives*from*a*single*mRNA* • product*of*one*gene* § only*contains*sequences*that*have*been*transcribed*into*RNA* § each*made*from*a*processed*mature*mRNA* • introns*removed*and*exons*spliced*together* § important*role*in*molecular*medicine* • important*proteins*synthesized*in*E*coli*from*genes*cloned*into* plasmids* o Expression*vectors:*plasmids*that*can*express*proteins*in*E* coli* § Contains* • Bacterial'promotor:*allows*cell*to*transcribe* cDNA* • RBS:*allows*mRNA*to*associate*with* ribosome* • Intrinsic'terminator:*halts*transcription* downstream*of*clone*gene* Constructing*a*library*from*genomic*DNA* o Steps* § Genomic*DNA*is*digested*by*a*restriction*enzyme*into*millions*of*discrete* fragments* § Ligate*fragments*into*vector* § Transformation* § Plate*cells*into*agar*plates* o Restriction*sites*don’t*have*a*relationship*to*the*structure*of*genes* § Single*gene*is*more*likely*to*be*clones*as*many*pieces* § Genomic*DNA*many*span*the*junction*between*neighboring*genes* Vast*majority*of*eukaryotic*transcripts*contain*at*least*1*intron* o cDNA*synthesized*from*mRNA* § introns*are*already*removed* Alternative*splicing* o If*pre-mRNA*undergoes*alt.*splicing,*RNA*sample*will*contain*isoforms*of* processed*mRNA* Open*reading*frame* o Initiation*codon*to*stop*codon* Experimental*techniques*to*determine*gene*product*expression* o Grind-and-Find* § Tissue*cells*à*homogenizer*à*homogenate*

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Tissue*sample*is*homogenized* Then*homogenate*is*probed*for*presence*of*specific* RNAs/proteins* Disadvantage:*gene*products*are*mixed*together*in*final*analysis*

• o Northern*Blot* § Population*of*RNAs*separated*by*gel*electrophoresis,*then*blotted*onto*a* physical*substrate*(nitrocellulose)* • Specific*RNA*detected*via*hybridizing*blot*with*compl.*nucleic*acid* probe*tagged*w/*a*label* o DNA*microarray* § Starts*with*a*cloned*DNA*library* § Microchip*–*solid*surface*with*DNA*placed*orderly* • Each*spot*has*tethered*copies*of*same*ssDNA*sequence* • Different*spots*have*different*genes*–*only*1*spot/gene* § To*probe*a*microarray,*mRNA*is*isolated*from*a*tissue*sample*and*used* to*synthesize*a*pool*of*fluorescently*labeled** • Fluorescent*label*=*#*of*cDNA*bound*to*it* § Pool*of*labeled*cDNA*à*hybridize*at*high*stringency*with*DNA*tethered* on*microchip* • Transcribed*at*high*rates*=*brighter* o Bright*=*greater*transcription* o Transcriptome:*entire*set*of*RNAs*being*expressed*by*a*cell/tissue/organism*in*a* given*time* o mRNA-sequencing* § measure*relative*#*of*each*transcript*in*sample* § steps* • collect*mRNA* • make*cDNA* • add*adapters*for*sequencing*primers* • sequence* • use*bioinformatics*to*align*sequences*and*count*transcript*levels* o In*situ*hybridization* § Method*for*visualizing*mRNA*expression*in*the*cells*where*it*was* expressed* § Steps* • 1.*Synthesize*a*labeled*nucleic*acid*probe*of*known*sequence* • 2.*Hybridize*probe*to*mRNA*in*a*population*of*a*fixed*and* permeable*cells* • 3*&*4.*Wash*out*un-hybridized*probe,*and*visualize*the*labeled* cells** § probes* • must*have*a*complementary*nucleotide*sequence**

mRNA*represents*“sense”*strand*and*complementary*probe*must* have*an*antisense*sequence* • probes*can*be*either*DNA*or*RNA*(preferred)* • can*be*labeled*with*a*variety*of*different*chemical*markers* o cells*à*homogenization*à*chemical*separation* § RNAs:*specific*RNAs*can*be*detected*by*hybridization'to'complementary' nucleic'acids' • Northern*blots*à*single*genes' • In*situ*à*single*genes' • DNA*microarrays*à*thousands*of*genes' • mRNAseq*à*thousands*of*genes' § Proteins:*specific*proteins*can*be*detected*with*antibodies** • Immunohistochemistry* o Western*blots:*similar*to*northern*blot*but*detects*protein* using*antibodies* o Immunostaining* •

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Lecture'24:'Overview'of'Gene'Regulation* DNA* o Storage*molecule* o Gene*expression*is*the*readout*of*info*through*synthesis*of*gene*products* o Gene*regulation:*mechanism*that*cell*uses*to*control*gene*expression* Constitutive*vs.*Facultative*Gene*Expression* o Constitutive* § Those*essential*for*cell*survival.,*aka*RNA*pol* § Similar*level*of*expression* o Facultative* § Many*genes*are*required*under*specific*conditions* • Genes*can*be*turned*on*or*off* • Only*expressed*when*cell*encounters*particular*environmental* signals* • Some*genes*are*only*expressed*during*specific*phases*of*the*life* cycle* Regulation*of*protein* o Steps* § Look*at*slide*10* o Controlling*gene*expression*at*the*level*of*transcription* § Prevent*synthesis*of*unwanted*RNA/protein* § Require*relatively*few*regulatory*molecules* Basal*transcription* o Simplest*form*of*transcription*regulation*in*bacteria* § RNA*pol*holoenzyme*recognizes*the*promoter*DNA*and*initiates* transcription*by*itself*

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Transcription*factors:*regulatory*proteins* o Sequence*specific*DNA*binding*proteins* § When*bound*to*DNA*they*can*influence*the*rate*at*which*pol.*initiates* transcription* o RNA*pol*binds*near*transcription*factors* § Operator*can*bind*to*LacI* • When*bound,*LacI*represses*transcription* § CAP*site*is*a*sequence*that*can*bind*to*CAP*protein* • When*bound,*CAP*activates*transcription* Electrophoretic*mobility-shift*assay*(EMSA)*aka*gel*shift*experiment* o Labeled*dsDNA*of*known*sequence*is*mixed*with*a*purified*protein*and*given* time*to*bind* § Doesn’t*bind*to*protein*à*DNA*migrates*as*a*single*band*of*predictable* size*(left*lane)* § Does*bind*to*protein*à*migrates*more*slowly*and*produces*a*second* shifted*band*(right*lane)*

***************************************************************** * - Structural*motifs*in*protein-DNA*interactions* o Helix-Turn-Helix*(HTH)/homeodomain* § Second*helix*–*holds*onto*DNA*backbone*and*positions*recognition*helix* in*major*groove* § Recognition*helix*–*inserts*into*the*major*groove*and*reads*base*pair* sequence* • Forms*covalent*bonds*with*chemical*groups*along*edges*of*paired* bases*when*its*finds*the*correct*bp* § Structural*motif:*secondary/tertiary*structure*shared*by*diff.*proteins* • Diff.*amino*acid*sequences*that*rec.*diff.*DNA*seq.* o Leucine*Zipper* § * o Helix-Loop-Helix*(HLH)* o Zinc*Finger* o Probability*that*a*binding*site*is*occupied*depends*on*

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Protein*concentration* Chemical*affinity* • Binding*strength*btw*protein*and*DNA*sequence* • La*Chetelier’s*principle* Genomic*DNA*of*a*cell*often*contains*multiple*binding*sites*for*the*same*transcription* factor*protein* o Optimal*LacI*binding*site* o O1*operator,*lac*operon* § 18/20*bp* o O2*operator,*lac*operon* § 14/20*bp* Allostery* o Regulation*of*a*protein’s*function*by*the*binding*of*an*effector*molecule*at*the* same*site*other*than*the*protein’s*active*site* o Cyclic*AMP*(cAMP)** § Used*as*an*intracellular*signaling*molecule* § Alters*DNA*binding*of*HTH*protein*CAP* • CAP*dimer*undergoes*a*conformational*change*that*rotates*its* rec.*helices*(R)*by*90*degrees* o R*must*be*parallel*to*one*another*à*fit'into'2'consecutive' turns'of'the'major'groove*

* Lecture'25:'Bacterial'Gene'Regulation:'the'lac'operon* -

E.*coli* o Metabolizes*to*obtain*carbon*and*energy* o Growth*=*most*rapid*and*energetically*efficient*when*using*GLUCOSE* § Glucose*=*abundant,*cell*minimizes*the*expression*of*enzymes*used*to* metabolize*other*organic*nutrients** • E*coli*has*a*5x*reduction*in*the*expression*of*lac*operon*gene* products* • No*lactose*=*lac*operon*is*strongly*repressed*(even*if*there’s* glucose)* o Lac*operon* § Expression*regulated*almost*entirely*at*level*of*transcription* § lacZ*àbeta*galactosidase* • enzyme*that*cleaves*disaccharide*lactose*into*glucose*and* galactose*for*further*metabolism* § lacY*à*lactose*permease* • membrane*transport*protein*that*allows*lactose*to*enter*cell* § lacA*à*thiogalactoside*transacetylase* • inactivates*certain*toxic*sugars*from*entering*the*cell*thru*lactose* permease* § transcription*and*repression*are*mutually*exclusive*states*

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lacI*gene*à*lac*repressor* o HTH*transcription*factor* o lacI*binds*to*specific*DNA*sequence*to*lacO*operon,*adjacent*to*lac*promoter** o binding*site*for*RNA*pol*&*and*LacI*repressor*=*very*close*that*only*1*can*bind*at* a*time* consensus*promoter*vs*lac*operon*promoter* o lac*operon* § weak*promoter*à*RNA*pol*holoenzyme*has*low*affinity*for*lac*promoter* o lacI*operon* § strong*affinity*for*lacO*operator* • lacI*binds*DNA*and*blocks*transcription*95%*of*the*time*

***************************************** * - Jacob*and*Monod’s*model*of*repression* o Lactose*absent*à*repressor*protein*binds*to*lacO*operator*DNA*à*NO* transcription* o Lactose*available*à*inducer*molecule*(L)*binds*to*lacI*protein,*altering* conformation*à*transcription*occurs* - Allolactose*–*lactose*isomer* o Physic...