Boston College Premed – The journey of a Boston College ...
Genetics and GenomicsForward geneticsPhenotype to genotypeReverse geneticsGenotype to phenotypeCell is the basic component of organismsNucleus contains the genesMitochondria have their own genomeProkaryotic cells differGenetic material in a nucleoid regionCell is organized but has no organellesAlmost everything is encoded in the DNADNA karyotype-lay out chromosomesCentromereHelps the chromosomes migrate from the middle of cell to polesMetacentric=middleSubmetacentric=below the middleTelocentic=at the endCell division is essential to lifeMitosis-division (exact copy)Meiosis-gametes (not an exact copy due to crossing over)Spermato/oogenesis2 separations to get haploid cellsCell must condense into chromatinSpindle attaches to kinetochore via the centromereDNA replication can induce errorsMutations or other changesIf it was perfect there would be no variationSource for variationDNA replication and repairCrossing over and chromosome segregationCell cycle is monitored by checkpointsG, S, and MG0= nondividing cellInterphase is G and SThe checkpoints can let mistakes throughThey check for DNA damage or a failure to replicateSomething is wrong=apoptosisPhenotypeAppearanceWhat is expressedCould be complexGenotypeWhat do the genes sayHomo/heterozygousDominant vs recessiveWT vs MutantWildtype is the normal that is definedMutant is any changesOnly 1 WT, but many mutantsMendelFound that in the F1 generation only one gene/phenotype dominatedBut if F2 it was a 3:1 phenotypic ratioChose phenotypes coded for by 1 geneMonohybrid crossOnly one gene being crossedStart with homozygous parental strainsRecessive allelesOnly expressed when two copies of the gene are presentIn most cases the WT is dominant, but WT can also be recessiveHomozygousTwo alleles the same Can be dominant or recessiveHeterozygousTwo alleles are differentDominant will be expressed in most casesHemizygousOnly one allele presentDihybrid crossTwo genes cross to see effect9:3:3:1 outcome in the F2 generationIndependent assortmentIndependent assortmentCombine the probability of one trait w/ probability of getting anotherMultiplyTest crossCan determine genotype if unknown but have a known phenotypeDifference between homo and heterozygousCross unknown with homo recessiveIf homo- get all dominant expressionIf hetero-get some recessive expression (1/2)Human crossesMultiple different disordersDominant diseasesRecessive diseasesPedigreesCan follow a disease in a familyCan determine its genotypeRecessive-skips generationsDominant- in all generationsX-linked=expressed in more males then femalesFemales carryExpressivity-the overall expression of the disease (how bad it is)Penetrance-not everyone gets the disease (have the gene but don’t express it)Out of the people who have the disease what % express itProbability and statistics of Mendelian geneticsP(A,B)=P(A) X P(B)P(A or B)= Pa +PbP(a/b)=Pa/PbBinomial theoremUsed to calculate the probability of any specific set of pairs of outcomes among a large # of potential eventsP=n!/s!t! X asbtS=# of a outcomesT=# of b outcomesChi-square analysisVariation between the observed and expectedSee if there is enough variation to reject the null hypothesis which states that nothing is happening (random chance)P must be less than 0.05 to reject the nullUse a graph of degrees of freedom (# of phenotypes-1) and x squared to determine PClasses of mutationsNull mutationDestroys the geneRemoves the allele completelyLoss of function mutationCould be nullDiminishes expression or function, or destroys a geneUsually recessive, need two mutations to allelesGain of function mutationSome mutation causes a new function Can change the phenotypeEx: flies with legs in their headDominant mutationsWhy is simple genetic dominance most often observes for geno/phenotype?Only need one allele present to function completelyMutationsMissensePoint mutation where the codon and changes the AA and proteinNeutralChanges the codon and AA but not the proteinSilentChanges the codon but not the AA or proteinNonsensePremature stop codonComplete dominanceHomo/heterozygous express the same phenotypeIncomplete dominanceHeterozygotes have an intermediate phenotypeCodominanceExpress both alleles in the heterozygoteEx: blood typeRecessive lethal mutationsThe homozygous recessive is lethal and will not surviveDo not factor it into the probabilities since they are unable to pass it onMixed modes of inheritance modify the 9331 ratioEpistasisThe effect of one gene depends on the presence of one or more modifier genesEx: agouti mice-can only get the agouti pattern if colored a certain colorRecessive or dominant epistasisNovel phenotypesGet something completely unexpected from a crossPleiotrophyOne mutation has a cascade of effects in the bodyComplementationHelps to determine where in the genome the gene is locatedIf in the same place, the cross leads to a mutationIf in different places the cross leads to a normal phenotypeSex linkedGenes located on the x chromosomeMales have to get their x from the mom and their y from the dadPedigreesAgain help see the expression patternDoes a genotype always result in the same phenotypeNo, because of penetrance and expressivityTemperature sensitive phenotypesHeat and cold sensitive mutations (conditional)See a level of expression changesLocation can also affect expressivityDNAFunctionsReplicationInformation storageInfo expressionVariation through mutationAllows new characteristics to evolveCentral DogmaDNATranscriptionRNATranslationProteinHas to flow in this direction unless a virus goes from RNA to DNA with reverse transcriptaseRibosome is formed by rRNAmRNA is loaded into the ribosometRNA brings AA to the ribosomesDNA and genome sizeMore genes doesn’t mean more complexitySuch thing as alternative splicingDNA as the genetic materialGriffith’s transformationFound that transformation occurred by some moleculeAvery, Macleod, McCarthyOnly when using DNAse did transformation not occurHershey-ChaseUsed bacteriophages and labeled moleculesDNA with phosphateProtein with SulferFound labeled DNA in the cellRNA can be the genetic materialViruses can have ss/ds DNA or RNAReverse transcriptaseIntegrationDiscovery of DNAX ray crystallography gave the idea of double helixDNA factsDNA is right-handed (right hand rule and thumb up)Every strand has a 5’ and 3’ endA is always bound to TG is always bound to CA + T + C + G = 1G3CA2TPhosphate connected to sugar, then the sugar is connected to a basePurines (Double Ring)G, APyrimidines (Single Ring)C, U, TSugar backboneRNA has an additional hydroxyl at the 2’ carbonDNA lacks the 2’ hydroxylWhen a sugar and base are bonded with phosphate=nucleotideWithout phosphate=nucleosideUp to 3 phosphate groupsDNA is made in the 5’ to 3’ directionWhy cant it be made in the other direction? Cant possibly add on to the phosphate group at the 5’ endPhosphate is negatively chargedIf together-will repel each otherNeed to make up the outsides, with the bases in the middleDNA has a negative chargeMigrates to the AnodeDNA’s densityG-C bond is more dense due to 3 H-BondsThe higher the G-C content the more dense the DNADifferent DNA melting points as a resultFISHTest to detect Nucleic AcidsDNA replicationSemi-conservativeEvidence=2 rounds of replication with labeled DNA strandsMany generations-only trace amounts of the old-mostly newBacterial ReplicationStarts at a single origin of replicationBidirectional replicationDNA Polym READS from 3’ to 5’ and SYNTHESIZES from 5’ to 3’ a new DNA strandDNA polymI, II, IIIAll can proofread and replace their mistakesHoloenzymesProtein machine made up of multiple proteins and TF’s BacteriaOrigin of replication is defined by a repeated sequence (9 mer)DNAa molecules bind and create an initial bubble of replicationDNAb/c bind to the bubble and initiate helical unwindingPrimase adds an RNA primerDNA polym startsLeading vs lagging strandAll replication proceeds towards the replication forkOne strand is continuousOne strand is discontinuousNeeds multiple primersMultiple okazaki fragmentsLigase sticks togetherDNA poly IReplaces the RNA primer with DNADNA gyraseUntangles the DNA helixSpeedEuk > Pro because there are multiple origin sitesEuk are not circularHave an issue with end of chromosomesSome cells have telomerase, which acts as an end primer to avoid losing some of the telomereMost cells don’t have telomerase and lose a small portion with each replicationTelomerase is only very active during large periods of replications, or when the cell is a stem cellReplication and recombinationNeed a single stranded break, then a ligation to a different placeCrosses with its homologous region and allows for recombination because a piece of DNA switched from one chromosome to anotherTranscriptionTranscriptome-all transcriptsProteome-all proteinsMetabolism-all metabolic compoundsTranscriptionHelp get an RNA messageNeed a template strand of DNA to get to RNARNA is identical to the coding strand, but is matched up with the template strandProkaryotic cellReplication, transcription, translation occur in the nucleus (nucleoid region)Need an RNA polymeraseScans for an RNA binding siteNeed the sigma subunit to recognize the specific initiation sequenceNascent RNATranscriptSigma factor dissociates after a few nucleotides of the RNA strand is built upOnly necessary for binding and recognizing the promotorRecognize TATA box upstreamOperonsGenes often found in a segment togetherGet a polycistronic mRNAOnly found in prokaryotesRibosomes translate as mRNA is being transcribedNo posttranslation modificationOccurs faster than in EukQuickly ramp up protein productionEukaryotesMany more regulation of the mRNASeparated into compartmentsRNA typesmRNAtRNArRNAmiRNAcatalytic RNAChromatin in EukDensely packed DNA and organized by histonesBefore transcription may need to modify the chromatinHetero/Euchromatin3 types of RNA polymeraseI=rRNAII= mRNA and snRNA (nucleoplasm)III=ssrRNA, tRNA (nucleoplasm)RNA Polym II promotors have a core promotor, and enhancer elementsTATA boxNot a lot have it, but if a gene has it, it is essential to transcriptionBinds the RNA polymerase after binding TATA Binding ProteinAllows for a transcription regulationBrings other RNA poly to site to increase regulationCAAT boxAnother example of a TATA like binding elementEnhancersSpecific sequence that can be located in front of, in, or after the geneIf located in the gene it keeps the gene from being translatedCan activate or depress depending on locationTFGeneralized proteins that bind specific sequences to regulate genes and expression levelTranscriptEukaryotes need to mature itAdd a methyl G cap and poly A tail to stabilizeAlternative splicingExons vs intronsIntrons spliced outImmature RNA s always longer than mature RNA (remove introns)ComplexityThink about number of proteins, not the number of genesGenes also interact with each other in different ways (regulate)SplicingGroup 1Make rRNANeed a guanine to bind to an active site within the intronExpressed hydroxyl, this attacks the donor site at the other end of the intron and splices it outGroup 2mRNAneeds snRNP’sget a complex that forms lariat loops that splice out intronsexons ligatedmodify the transcriptRNA editingSubstitution editingGet a change of a nucleotide in a transcriptTwo forms of a protein depending on editingInsertion/deletion editingCan alter the function and shape of proteinOr bring proteins into the proper reading frame to establish functionTranslationThe codon tableCode for an AAStart codon AUG begins the open reading frameErrors:Spontaneous mutations lead to base pair changesPoint mutations: changes a proteinFrameshift- changes many AA and proteinLength=basepairs/3Weight=aaX110 daltonsThe triplet code is nearly universal and can mostly use the same tableIn viruses, they overlap in viruses to save spaceDifferent messages within same transcriptSingle mutation can affect multiple genesTranslation of mRNA occurs only when ribosomes and tRNA are present and functionaltRNA=clover shapeh-bond to the complementary AAribosomeprokaryotes70s ribosomesEukaryotes80s ribosomesChanging tRNA’s with AA’sNeed an empty tRNAAA synthetase puts the AA on the tRNANeed ATP energyActivated enzyme complex (AA, AMP, aminoacyl tRNA) attaches the AA to the tRNAFactors associated with 3 different phases of translationInitiationElongationTerminationRibosome is not formed until the mRNA binds the small subunitThen the large subunit binds3 ribosomal sitesAminoacyl site=AA sits in the tRNAPeptide=growing peptide chainExitStop codon causes the complex to fall apartReleases the peptideMultiple translational complexes form on a single mRNAAmino AcidsR Group only thing that changesHydrophilic/hydrophobicPolar (charged)The r group differs in forl/functionChange the folding by mutationsProtein sequencePrimary=AA sequenceSecondary=alpha helix or beta sheet (H-Bond stabilized)Tertiary=whole protein foldingQuaternary=multiple proteins foldingDomain-functional part of protein that has a certain structurePost protein modifications (post-translational)N terminal AA is often modifiedAdd carbs to the proteinGolgi editingFunctions of proteinsStructuralContractileSignalingStorageTransportEnzymaticRolesEnzymaticLower activation barrierSignal sequence-domain that attracts substrateMembrane anchoringMutationsGermline vs somaMuch more dangerous in germline, passed onto future generationsClasses= LOF, GOF, null TransitionPurine changed into a different purineTransversionPurine changed for pyrimidineRepeat expansionContinue to get repeated sequencesGenetic analysisUse mutations to ID mutations and their resultant phenotyoesInduce many mutations to get a specific mutationOriginProofreading errorsDNA replicationBut you do get a lot of repair of these mutationsTautomeric shiftOne H switches position within nucleotideLeads to mispairing and replication errorsT to G and C to AWhen replicated back to their normal binding partner, causes mutationDeaminationAmino group in C or A converted to a keto group, which changes the basepairingDepurinationLose a nucleotide within the DNAOxidative damageOxygen damages the DNATransposonsPieces of DNA that can insert or move within the genomeReplication slippageMultiple repeatsGet an increased # of copy number variantsBase AnalogsIncorporates a different nucleic acid5 bromouracil (binds to A)Alkylation Donate methyl or ethyl groups to amino or keto groupsGuanine to 6-ethylguanineUV radiationThymine dimersRepaired by nucleotide excision repairAccessing genotoxicityBefore anything is released used the Ames TestHave a control side and get the number of random background mutationsAdd the mutagen, see if any difference than the background rateRepairDNA polymerase can proofreadMismatch repairMut S/L/and H scan the DNA for the incorrect base pairsThen stick on the DNA and recruit DNA polymeraseExcision repair (during DNA replication)DNA polymerase finds a lesion, it skips overREC-A comes back and fills in the gapDNA ligase ligates it togetherSOS repairLast resortInduces more mutationsOnly occurs when there is massive mutationPhotoreactivation repairDimer formsDimer repairedNormal pairing restoredBase excision repairRecognizes a single wrong nucleotideBase removed by DNA glycosolaseAP endonuclease recognizes lesion and nicks DNADNA polymerase fills gapDouble stranded break repairMultiple lesions makes DNA unstableActivated during late S/early G2 stageWhen sister chromatids are available to serve as templatesEvolutionary GeneticsDarwinian evolutionSpecies have a common ancestorNeodarwinismDiscovery of geneticsEvolution requires:Variation between organismsCompetition between individualsSelectionDescent from common ancestorsCan use genetics to find these relationshipsTwo formsMicro/macroevolutionLarge and small scalePhylogenetic tree-shows relationship between speciesStasic-doesn’t changeAnagenesis-one species evolved into a different oneCladogenesis-species diverged into 2 separate onesMorphologySpecies based on the way they look?Not a great model due to different looking organisms being of the same speciesBiological species conceptDefine a species based on the ability to reproduce and have offspringSelection and fitnessAdvantage for one characteristicGet some fitness affectFitness-measure in the success of breedingMutations usually aren’t goodHowever may be advantageousSelected forStabilizing selectionLess genetic variationWhen the environment is stableDirectional selectionShift towards one sideWhen the environment changesDisruptive selectionEnvironment heterogeneous Can harbor two different organismsMaintain genetic variationVariation is not limitedSequence the genome to see the differencesChange environment, some mutations become advantageous and are selected forCost of variationProtective effects of sickle cell anemia against malariaFitness to genotype changes with the environmentSpeciationPre/postzygotic barriersEx: geographical separationPopulation geneticsHardy WeinbergDescribes an ideal population’s allele and genotype frequenciesP2+2pg+q2=1P+q=1Can predict what will happen in the next generation if no natural selection occursStronger selection against the recessive allele if homo recessive is fatalCan be small or weak selection against an allele (or large)Just mutationsTakes many years for mutations to become a part of the species unless the environment changesGenetic driftThe changes in allele frequencies due to chanceMore of an effect in a smaller populationFounder effectWhen a new population is started due to migrationWill not have the same allele frequencies as beforeInbreedingInbreeding depression (lose heterozygotes)No new influx of genetic materialF valueF=1 all homozygousF=0 no inbreedingDistance apart in years=# of mutations X mutation rateDNA organizationSimple chromosomesViral and bacterial chromosomes often consist of single DNA moleculesBacteriophage=lambda (lollipop head)Circular replicationCut bu a nucleaseCopied discontinuously and continuouslyBacterial DNA packagingEcoli supercoils the DNADNA has no tension due to turnsEukaryotesOrganize using histone proteinsCondensed state get G-bands (dark and light)Can alter the packaging to get to genesDNA loops out of chromosomes when neededNucleosomeHistone octamerSolenoidGroup of 6 nucleosomesLooped domainsChromatin fiberChromatidNet packing ratio of 500:1Repetitive DNA98% is repetitive DNACentromeresSister chromatid cohesionAssembly site for kinetochoreCENThe minimal DNA required for centromere functionSatellite DNARepetitive pieces of 2 or 3 nucleotides that are constantly repeatedDNA isolationSatellite DNA has a lower densityLess dense with more A-T bondsVNTRVariable number of tandem repeatsSTRShort tandem repeatsVery short 5 or less basesLINELong interspersed nuclear elements (transposon)SINEShort “ “Ribosomal genesRepeated in the DNAEpigeneticsHistone modificationCan be passed onReversibleEpigenatorsEnvironmental signals (internal or external)Signal is transduced to the cellHistone modificationHistones have a tail that can be modifiedAcetylationOpens upDeacetylation closesMethylationOpens or closes depending on locationHDACHistone deacetylation complexCloses the DNA upHATHistone acetylation complexOpens dna up CPG islandsSites where the DNA is methylatedImprintingIGF2 not turned offHypo/hyper methylationEpigenetic inheritance can lead to cancerVariation in chromosome number and arrangementKaryotypeGroup chromosomes and banding patternsAneuploidy2n±x chromosomesEuploidyMultiples of n chromosomesPolyploidyMultiples of the same geneAuto/allopolyploidyAuto=duplication of whole genomeAllo=duplication of 2 diff speciesNondisjunctionDoesn’t separateLeads to trisomyTrisomy 21=downsTrisomy 13=patauTrisomy 18=EdwardsChromosomal rearrangementsNeed breakage of a chromosomeTerminal deletion (lose piece at origin)Intecalary deletionForm a deletion look and it ejects a gene out of a chromosomeDeletion loopDuplicationUnequal crossover between 2 sets of homologous chromosomesrRNA present in many copiesCNV (copy # variants)Chunks of repeated DNA in chromosomes due to duplicationCan be present within promotor regionsCan cause an increase in the replication of the geneInversionMay express new genesCan happen due to loopForms a 4 part breakageParacentricDoesn’t change arm lengthPericentricChanges the length of the armsConsequences during chromosomal inversionInversion heterozygote=one inverted and ore normal chromosomeCrossing over leads to nonfunctional chromosomesNonreciprocal translocationOne chromosomes steals from anotherReciprocalThey share-just changes chromosomesForms a cruciform tetrad during meiosisRobertsonian translocationExchange of small arm of one chromosome for the large arm of anotherCan get familial downsFragile XPieces of the X can break off at the endIts so thin because the DNA isn’t as condensedMicrobial geneticsLag, log, then stationary phasesAuxotrophs-cant produce certain compounds and need it to be addedGrow bacteria on selective mediaOnly grow with additions2 life cyclesLyticPhage DNA is injected into the cellCell begins to produce phage componentsCell lyses and releases phagesLysogenicDNA integrated into the hostDormantAll subsequent cells have viral DNAEventually when stressed, the cell produces virusesU tube experimentsJust the medium allowed to passConjugationNeeds a sex pilus and attachment between cells to pass the DNAAlso need a plasmid F factorWill not happen in U tubeHFR cellsHave the F gene in the DNA itselfWill conjugate but will not pass on the F gene to the other cellR factor encodes for antibiotic resistanceHorizontal gene transferWithin one generationVertical gene transferInherit from generation to generationF factor can integrate into the genomeThen it’s the HFR cellTransformationTake up free genomic DNA from the environment and incorporate itWill occur in U tube experimentsTransductionThe DNA is inserted via bacteriophage into another cellWill occur in U tube experimentsGenetic mappingUse recombination between the regions to map for mutationsDeletion mapping-map consequencesRecombination mapping-based on genetic exchangeLinkageTwo genes on a single pair of homologsNo exchange occursDistance matters in recombinationCount the recombinants and parentalMap distance=REC/(total) X 1001cm= 1% recombination observedTwo and three point mappingConsider single and double crossoversDouble cross oversFrequency is the product of the two SCO’sTableThe highest #’s are the parental strainsThe lowest numbers are the double crossoversOrder of the genes is based off of which one is in the middleFor the double crossover, the one that appears to change is the middleC=coefficient of coincidenceDCO observed/expectedInterference=1-CNever the expected due to one crossover inhibiting a secondSomatic cell hybridizationLinkage mappingSister chromatid exchangesDon’t see phenotype exchangesExactly the same genes but cant see changes without mutationsGWAS (Genome wide association study)The goal is to map phenotypes and where they appear on chromosomes based on mapsExtranuclear inheritanceInheritance of genes that are not contained in the nucleusChloroplasts and mitochondriaBoth only inherited from motherChloroplasts inherited from the MT+ parentMitochondrial inheritance can be tested with coloniesPetite colonies indicate something is wrong with mitochondriaSegregational=nuclear (1/2 petite)Neutral=cytoplasmic (all normal)Supressive=cytoplasmic (1/2 petite)ChloroplastsLarger DNA than mitochondria (more introns)MtDNASmallerGoes missingNo intronsThe origin of mitochondria is via the endosymbiosis theoryNuclear contributions to the mitochondria and chloroplastsVia nuclear genesPassed on via regular geneticsMitochondrial diseasesMERRF, LHON, KSSGenetic elements + virusesIS elements (bacteria)Insertion sequenceDefined by inverted terminal repeatsFlanked on both sides of the geneTransposonsRecognizes inverted terminal sequences specific for an ISInserts the sequence somewhere else in the genomeDNA bases transposon elements (tn)Can be largerHeteroduplexThe complementary sequence that helps it bud offIn the presence of Ac, Ds is not transposableBut Ac alone can transposeAc must still have its transposase geneDs lost its transposase functionBut still have the inverted sequencesNonreplicative/replicative transposonsRearrangements are mediated by pairs of tnsDeletion between two transposonsGet crossovers between repeatsGet circular deletionSeparate from chromosomeRNA based TE’sRetrovirusLTR=attracts RNA polymerase to make its productsLINESINEReplication (copy elements)Transcribed into RNA and proteinCan silence the transposon DNA Target for destructionRetrovirusIntegraseMediates integration of DNA into genomeRetroviral integrationssRNA to dsDNAreverse transcriptase cant proofreadretroviral buddingproducts packaged and moved to the PMDNA virusesHave a lytic/lysogenic life cycleRNA virusRemain RNA alwaysCan be + or – stranded+=no rdrp (translated directly)-=rdrp to transcribe to + strandRDRP=RNA dependent RNA polymeraseZoonoses=movement of virus from animal to humanRecombinant DNACut a plasmid vector with restriction enzyme (vector)Cloned DNA is cut with same REThen the two pieces of DNA get linked togetherThen introduced to host cells via transformationSelect cells with recombinant DNA by antibiotic resistance selectionLibrariesCollections of clonesCDNA library (complementary DNA)Higher complexity means the more coverageNeed 5 times the number of clones to cover a whole genomeMake sure all overlapsVectorsPlasmid vectors have a small amount of DNAPhage/cosmid vectors are largerArtificial chromosomesExpression vectorsShuttle vectorsRestriction EnzymesEndonucleases with a specific recognition restriction site where it cuts DNALeaves sticky endsCut every 4N base pairsN is number of bases in RE recognition sitecDNAget ds cDNA with reverse transcriptase and mRNAneed a selective marker in the vectorscreen for the vectorsPCR95=denature50=annealing75=polymerizationAmplify the DNA experimentallyReal time PCRProbe on the templateSee the florescence levelRestriction mappingCut the DNA with different enzymes and see how the DNA is put togetherSouthern Blot=DNANorthern Blot= RNAGenomicsSanger sequencingAble to do short segments of the genome (about 1000)Next gen sequencingSequences the entire genomeClone by clone sequencingCut up the genome into pieces using RE’sSmaller and smaller piecesThen insert into a large plamidYAC/BACFit together with overlapping clonesShotgun based sequencingUse different RE’s to cutSequence contigs (next gen sequencing)Overlap contigs using a computer systemRepetitive DNA is hard to overlapGene modelsID the UtR, initiation site, promoter, regulator elements, introns, and exonsSequence the cDNA or RNA so that you know what is expressed in a mature cellCan also get different mRNA based on alternative splicingDetermine the expressed pieces of a genomeComputer reads all three framesBest when there are no intronsDatabasesBLASTUses an algorithm to see how close a protein overlaps with the alignment of known proteinsDetermine % overlapAlso can determine functional domainsHuman Genome Project (HGP)20,000 genes3 billion base pairs98% noncodingThe noncoding DNA may have a regulatory functionMade partial chromosomal mapsGenes clusterDeserts in between genesDisease mapsMap the genes that cause diseases and where it is located on the chromosomeENCODELook at hetero/euchromatin and changes from cell/cellShows where the genes are going to be expressedCHIPChromatin immunoprecipitatetag the protein with antibodies to find the protein of interestOmicsvariation in genome size and gene number in bacterial genomesnot sure what the minimum # of genes is to make an organismOrganization is circular or linearEukaryotesLess gene dense (have introns)More DNAMore noncoding and repetitive portionsDogs used to compare to humansHave similar genesChromosome 15 responsible for the size of dogsLinkage disequilibriumTendency of two alleles to remain linked through meiosisSynthany=tells how similar genes are in terms of the order of genesComparative genomicsGenes can evolve based on exon duplication and exon shufflingCan trace the domains backAlpha and beta globulinSimilar on the molecular levelMake up a subfamily of proteinsMultigene familyEvolve by deplication and divergencePhylogenetic treeConservation of gene structureSuperfamilyAll the related proteinsGlobal Ocean Sampling (GOS)MetagenomicsLooked at the spectrumLook at everything as a whole and draw conclusionsToo large to look individuallyHuman Microbiome Project (HMP)TranscriptomicsLook at all the transcripts to see what is expressedAnalyze the transcriptsMicroarrays-specificChecks for a small sequenceChecks a lot of different small sequencesVery good and cheaper to check for cancerRNA sequencing= everythingSequence all the RNATypify diseases based on the RNA expressionChecking for cancer questionCheck for the DNA having the codeDNA microarray specific polymorphism (change)Full sequence if looking for cnv, or other repeatsTranscribed to RNARNA seqTranslated to proteinWestern blotMicroarrayLooks at a small subset of the genes or RNA and not the whole cellProteomicsLook at the proteins present in the cellWestern blottingCut proteins into pieces using trypsnUse mass spec to separate based on mass to charge ratioSystems biologyMake sense of all the dataConnect concepts and networksHow the pathways interact with one anotherHuman GeneticsFemales XX (homogametic)Males XY (heterogametic)Theory-the embryo can develop into male/femaleAt one point important changes due to the presence of the Y chromosome set the male into actionY chromosomePAR region (pseudo autosomal region)-allows the Y chromosome to pair with the X for mitosis and meiosisMSY region (male specific)-genes that make a male a maleSRY region (sex determining region)-induces the development of testesExpressed 6-8 weeks into developmentTDF=testes determining factorX inactivationDue to dosage compensationCreates a barr bodyUtilizes the Xic region and the T-six geneSingle Nucleotide polymorphisms (SNPs)Differences in genomes between organisms, or genetic variationGenomic variation (types of SNPs)RFLPRestriction fragment length polymorphismsOne of the first ways to distinguish between genomesAppearance/disappearance of specific restriction sitesVNTRVariable # of tandem repeatsThe more repeats, the earlier disease onsetsSNP’sSingle mutations at a specific locationCNVCopy number variantsLarge piece of DNA repeatedGWAS databaseLinks phenotypes to genotypesAssociates SNP’s with genomesNeed to adjust the p value when you have such a large sample sizeBonferroni-corrected significance cutoffOriginal p / N (sample size)PharmacogenomicsTry to associate peoples genomes to the way a drug functionsResponsivenessThe % of effectivenessDetermined by the genomeDrug ex: HerceptionNeed to sequence first, using microarrays to determine if the expression correlates to the diseaseCan only use if specific HER-2 mutationPersonalized medicineBased on a person’s genomeAdverse drug reactionsCost billions of dollarsPeople process drugs in different waysUltrarapid metabolizer > extensive metabolizer (normal) > Poor metabolizersProkaryotic gene regulationOperonsThe idea of an operon is that in prokaryotes, many genes that are expressed together are under the control of the same promoter elementsInducible operons (also known as adaptive, facultative)Only expressed when necessarySystem can be turned on/off depending on environmental stimuliPositive controlInducer in the system that turns on gene expressionNegative controlGenes that are normally on get shut off by the presence of the moleculeConstitutively activeAlways onLac operon (inducible)Cis acting regulatory sites are present upstream of gene clusters3 genesLacZ=B-galactosidaseLactose to glucose and galactoseLacY=lactose permeaseFacilitates entry of lactose into the cellLacA=lactose transacetylaseDetoxifying enzymeRepressionLac IExpressed and binds to the operator site to stop transcriptionPolycistronic RNA is created after transcriptionRepression of the Lac operonLacI repressed when presentBinds the operator regonOnly leaves when lac is present and binds to the repressor (and glucose is absent)MutationsLacI mutantsCant bind to the promotorStays on constantlyLacI mutantsCan bind to the promoter but not lac, so always offOperator regionWont bind the repressor-always onKnown as the Oc mutation because it is constitutively activeMake diploids to see mutation effects (Merodiploids)The operator needs to be in front of the genesSo if a mutated operator is in the plasmid, will not have an effectRepressor can be made anywhere and travel to bind the promoterIPTG can induce the lac operon expreeionGlucose is the preferred carbon sourceLess energy cost to the cellGlucose levels high, cAMP levels lowcAMP levels are high when no glucosecAMP binds to CAP (catobolite activating protein)CAP induces expression of the lac operon (assuming lac is present)Lac repressorHomotetramerInserts 4 O sequences that then are pulled together to form a repression loop and stop transcriptionTrp operonRepressible systemOpposite of lacThe presence of trp shuts off the operonThe lack of trp turns it onThe repressor is bound to the operon when it is bound to trpTrp mutantstrpR mutantsalways on trpO mutantsalways on because it cant be blockedtrpP mutantsalways offattenuationan interaction between transcription and translation that regulates expressionleader region is in front of the trp operontranscribed onto the mRNA and has a regulatory functiontrp present=terminator hairpin and no transcriptiontrp absent=anti-terminator hairpin and transcriptioncharged tRNA’s determine if trp is present or not if charged tRNA present-there is trp present mediated by trp RNA binding attenuating protein (TRAP)TRAP enables formation of the transcription terminator hairpin if it binds to enough trpANTI-TRAPNo binding of trp, forms the antiterminator hairpin loopArabinose operonUnder both inducible and repressible control3 genes and a CAP binding site in the E.coliBoth types of control are mediated by Ara CDon’t invest in the synthesis of any other sugar if glucose is presentEukaryotic gene expressionDomains separate chromosomesChromosome territoriesInterchromosomal domains between chromosomesCompactness of DNAAcetylation and methylation of histones and DNA regulate the compactness of chromatinChromatin remodeling complex (swi/snf)Opens up the DNANeeds ATP to remodelMoves the nucleosomes apartLess tightly wound makes the DNA more accessible to transcriptionInsulator elements prevent the spread of chromatin remodelingCis acting sites in chromosomal DNA bind to transcriptional regulatory proteinsPromotersEnhancersSilencersPromotersFocusedAlways initiates transcription from the same siteDispersedInitiates transcription from multiple sitesGet multiple transcriptsFocused promoter elementsBREB recognition elements-affect complex bindingTATAINRMTEMotive 10 elements-help RNA polym bindDPEDownstream promoter elements-help RNA polym bindCAAT boxRequired for initiationGC boxBinds TF’sEffect of mutationsMutate promoter elements-reduce the transcription levelCis acting elements bind TF’sTF’s often expressed in time and tissue specific patterns and can recruit or interact with RNA polymerase, and other Tf’s, and respressor proteinsBasal transcription level vs induced transcription levelFunctional domains of TF’sCan screen the genome and ID the TF’s based on their propertiesDNA binding domainsHelix turn helixTrans-activated domains (repressors)Zinc finger DNA binding domainBasic leucine zipperAssembly of TF’s Ex: RNA polymeraseTBP (Tata Binding Protein)Binds to the sequence and brings in TAF (TATA associated factors)Polymerase comes in and forms the complexTBP and TAF stays in place to recruit additional transcription complexesEnhancersMore upstreamHelp attract TF’sCan affect how fast a complex is madeIncrease the rate of DNA unwinding and RNA polymerase release from the promoter to initiate transcriptionEx: UASgConstitutively active post translational regulationalternative splicingex: sex determination in DrosophilaSLX gene is only active in femalesGet female only splicing that leads to the production of the DSX-F proteinDSX-M protein present in malesmRNA stability controlcontrol the half life of the mRNAdepends on the transcription rate, processing, and degredationProtein levelAutoregulationEx: tubulin subunits bind to the growing polypeptide chainCan stall the translationGet RNAse to degrade the mRNAIron regulationRegulates the ferrin geneNo translation if an Iron regulatory protein is bound (which means no iron is in the cell since iron binds to release it)Too much iron?Binds to IRP, which down-regulates the mRNA (which is only stable when IRP is bound to it)miRNA and siRNAboth bind to the RICS and RITS complexescreated from dsRNA via the dicer proteinRISCDegradation of mRNA complementary to the sequence of the small RNADownregulates the mRNA that is not exactly complementary but closeRITSGoes directly into the cell nucleus and downregulates the production of the gene directlyGene FunctionForward geneticsGenome wide genetic screens for mutants with specific phenotypesId the genotype that creates the phenotypeReverse geneticsDefine every gene in the genome based on sequence analysesReduce/eliminate functions of specific genes and assess the phenotypic impactsModel organismsEasy to growShort generationAbundant progenyCan cross in large numbersYeastSimplest eukaryoteHaploid and diploid alternating generationsPhenotypes are evident in haploidDiploid allows for recessive lethal mutations to be studiedDrosophilaNo meiotic crossing over in malesDiploidRecessive lethal mutations are maintained in strains heterozygous for balancer chromosomesP-ElementsDNA transposons that insert into the genomeCan enable transformationWild type or altered copy of the gene to assess transgene functionReporter gene in which enhancer/promoter drives expression of beta-gal or other detectible genesNeed a positive selective markerCan use this to destroy genesRandomly inserts itself into the open reading frameP elements either insert or destroy geneMiceGenomic synteny with humansLarge scale genomic screens difficultCreating transgenics and gene knockouts/replacements is more feasibleMutagenizationMutagenize parental strain, then perform crosses to generate progeny that can be assessed for phenotypes of interestTypes of mutationsChemicalEMS, ENURadiationX-Rays, gamma radiationScreen mutationsGenetic screen helps select out the ones which were mutatedCan look at yeast and determine the stages of cell cycleSee any arrested developmentWill not grow if mutatedReplica platingGet the same colony and grow under different stressors to see if mutations are sensitive or if new mutations appear under stressScreening mutants (Balancer Chromosomes)Can screen for recessive mutations in diploids by creating a collection of mutagenized chromosomes in balanced heterozygotesAssess the phenotypic impact of homo/hemizygocityRetain mutations of interestUntangling pathsThe order of generation is based on epistasisThe effect of mutation in one gene masks or modifies the mutation in another genePathways affected by this-because every gene must be present to make a productUse epistasis analysis to determine which gene is at the top and which is at the bottomCan determine pathway order with mutation to genesScreens for suppressor mutations can ID additional genes in a pathway not Id’ed in an initial screen (second round of mutagenesis)Second mutation by chance mutates the other one t try and bypass the initial mutationModify the original phenotypeSuppressor mutantsDiminish/eliminate the phenotype caused by the initial mutationGene product sequenceMay reveal gene functionPresence of domains known to have specific functionsGene product functionInvestigated further using molecular genetic tools and techniquesMany different methodsTake gene see proteinTake protein see functionGene knockout/replacementWhere protein is expressed and what its function isCan ID the cDNA responsible for protein via libraryUse antibodies against the protein of interest to screen expression vectors/librariesExpressed clone contains sequences for the gene of interestCloning genes by complementation of genetic defects in heterologous or homologous cell/cell linesCan recover the function with human gene inserted into the yeastAssociate with the functionGene expressionWant to ID where and what the gene is doing in the cellsPlace and time of gene expressionTagged immunochemistry/florescence to see where and when the protein is expressedMiceSelection for insertion of positive selectable marker disrupting the target geneGet recombinant and negative selection against nonhomologous insertionsIntroduce knockout isolated cells into blastocystGet a chimera mouseNeed the chimera to get the homozygous line after getting heterozygous knockoutsChip-Chip SequencingAssess epigenetic stateRNAiRan interferenceHomologous RNAGet the RISC complex to degrade the target RNA leading to gene knockoutBioengineeringtransgenic pigs engineered to express green florescence protein (GFP)genetically engineered biopharm. Productscell lines genetically engineered to produce a medicine/drugbiologics produced using bacteria, fungi, and cell lines as bioreactorsCan create insulin for exampleExtract the A and B proteins from different cells and combine them to form fully functional insulinBiopharmingUse of GMP for production of biologicsBiologicsGenetically engineered biopharm productsExpression in bacteria, yeast, and mammalian cellsVaccinesEither inactive or attenuated sample of a virusCan be edible or injectedSubunit vaccineOne or more surface proteins from a pathogenGet an immune responseInject proteins into a person-still get the immune responsePlant genetic engineeringHigher yieldsDrought preventionStarted from artificial selectionSelect the best ones and breed themEPSPImportant to produce aeromatic DNAWe don’t make some of these AA’sTyrosine, threonineBacteria and plants make themDestroy operation of aeromatic AA’s so that the plant diesPut a strong promoter in to get a high EPSP synthaseLocating animals for production of biologics and protection against mastitis (staph)GM lysostaphin production cleaves the cell wall of the proteinUse florescence to detect thingsConstitutively on promoter turns on when the object is presentSynthetic bioWhat is the minimum genomeCan then begin to incorporate other thingsFetal karyotyping and genotypingAmniocentesisStick a needle in and take amniotic cellsChorionic villus samplingTake a sample from the placentaFetal cell sortingBlood sample from mother (some fetal cells)Helps get a karyotype and genotype on fetal cellsPreimplantation diagnosisPCR amplifying DNARFLPDetect 5-10% of genome wide sequence variationAso testing (allele specific oligonucleotide)Short oligonucleotide of defined sequence based on SNP’s hybridization PCR amplification of genomic DNA from sampleArray based genotypingMicroarrayLook for gene expression and levelcheck for SNP’s/CNV variationp53 genechipany of the 500 mutations that could lead to cancercan see the genes required for infection, propagation, and pathogenesiscan see which genes are involved in fighting virusesgene therapy for people with SCIDsonly a one gene fixnever officially provenMMLV virus used to insert the correct geneVirus that effects once and shuts downMajority of delivery vesicles are virusesRandomly integrate-so need better controlConcernsCapacity only 8kbCould provoke an immune responseBody PlanDevelopmental geneticsGenetic and molecular mechanisms underlying cellular and organismal development, homeostasis, aging and senescenceDevelopmentDevelop tissuesDeath of specific tissuesBalance between growth and deathSpecificationWhen genetic and positional cues confer a spatially discrete ID on cellsDeterminationCells time when a specific developmental state becomes fixedDifferentiationProcess by which a cell achieves its final form and functionHypothesisDevelopment-attainment of a different state by all somatic cells in an organismVariable gene activity hypothesisDifferential expression and action of genesControls developmentWhen and where are genes expressed and activeHow is gene expression regulatedPreformationSperm had little human inside that became biggerFertilization occurs when an egg and sperm fuse Maternal cytoplasmic componentsmRNA and proteinsfirst components to trigger developmentwithout these nothing would happenbody planvery similar in organisms within the same speciesPattern of organization-characteristics and recognizable traitsPattern formationAspects of development of the body planLeads to genesis of patterns or structures that make up the body planNumber of axes (primary)AnteriorPosteriorDorsalVentralAnimal body plans are segmentedThe body plan has 11 segmentsOften has appendagesDrosophilaHomologies among embryonic, larval, and adult body plansGoverned by a set of genesDifferent segments develop into different partsSegmental organization of embryonic and adult tissues is homologousSegmental disks develop into extremitiesImaginal discs rise to external structuresMutations that alter the body plan affect the pattern formation3rd segment develops into a second segment-fly has 2 sets of wingsEmbryogenesis over 24 hours get the body plan and imaginal discsSyncytial blastoderm (multiple nuclei)Followed by nuclear migration and cellularizationThe pole cells form at the posterior and are the precursors to a germ cell lineMaternal functions direct the AP and DV axesZygotic genesPart of the genome but regulated by maternal effect genesGap genes, pair rule genes, and segmental polarity genes form the body planThen homeotic genes (HOX genes) determine the fate of cells and specify the type of cell they will becomeNuslein-Volhard and WieschausDetermined which genes are important to body planMaternal effect genesForm the anterior posterior gradiantsGap genes are triggered (they are TF’s)Trigger certain genes in gap genes to form the band regionsFormation of discrete bands triggers pair rule genesDivide gap gene bands into smaller regionsActivation of pair rule genes activate segment polarity genesEven more dividedThen the hox genes are activated and specify the ID of each segment Gap genes are zinc finger TF’sActivate the next set of genesPair rule genesOften encode helix turn helix TF’sOverlap of TF’s or non overlap specifies specific segmentsMutationsRunt (mutated RunX2 protein) encodes a TFMouse doesn’t have proper muscle/bone developmentHumans get cleidocranial dysplasiaAutosomal dominant diseasesTwo Hox genes clusters in drosophilaAntennapedia complex and Bithorax complexHox genes and TF’s with HomeoboxDNA binding homeodomainDifferent complexes influence the further specification of segments into specific cell typesGene organization with Hox genesHox genes have a logical order in the DNANot intermixedCollinear with expression patterns in the embryoHumans4 human hox gene clusters39 totalControl A-P patterning in humans (and other vertebrates)5’ end genes Limb developmentHumans don’t get mutated very often (need a double mutation since diploid)get smaller changes (ex: polydactyly)more complex developmentsignaling pathwayscell signaling/signal transductioncentral to developmentwnt path, TGF-B path, hedgehog path, RTK path, notch signal pathcell signaling paths determine cell fatedepends on cell/cell interactions-need to talk to each othermediated by ligands and receptorsnotch signal path exampleMembrane bound ligand stimulates membrane spanning receptor, a portion of the receptor is transported to the nucleus and affects target gene expressionHuman NOTCH 1-4 mutationsAlagille syndromeLymphoblastic leukemiaSpondylocostal dysnstosisDevelopmental program initiated by NCID going into the nucleolus (after the delta protein binds notch receptor)Activates TF’sNotch in C elegans959 cells exactlyKnow exactly how all cells formStarts with the zygote then splits into adult over series of stepsAll divisions occur by specific developmental stagesUterineDevelopment is random between 2 cellsLag 2=signals Lin12 (receptor)Both cells expressing proteinsBy chance one will express more signal than the other (more Lag 2)This inhibits Lag 2 in the other cell and get more receptor in that cellMore signal= anchor cellMore receptor= ventral uterine precurson cellOnce the anchor cell is determined it increased production of Lin3Cells closest to the anchor get the most lin3Those cells are primary development cells (into the vulva)Cells around that, secondary development cellsno signal from the anchor-develop into skin cellsdepends not only on maternal factors, but other factors as wellhelp from gradients that trigger specific developmental programscells removed during developmentdon’t stop dividing-cancerproduced cells need to be removedoccurs in c elegans to get to the 959 cellshermaphrodites 131/1090 cells diemales 147/1178 cells die15 cell death (ced) genesApoptosis=cell death (programmed)Ced9 expressed, shuts down ced3-4 and the cell survivesVice versa, no ced 9, get cell deathGain of function mutation in ced9- leads to no cell deathBCL-2 is the human version of ced9Overexpression prevents cell deathCancer genomicsDisease of somatic cells25-33% of the human population is affectedKept in check y autoimmune surveillance and cell deathSomatic cell dysfunction is due to dysregulation of cell growth and movementUncontrolled cell division and the avoidance of cell deathNo apoptosisDysfunctionsProliferation-excess cell growthMetastasis-movement of cancer cellsBenign tumor- local mass of cellsMalignant tumor-cells metastasize and the tumor has access to a blood supplyPrimary tumor-the initial siteSecondary tumor-the site where the tumor spreads toGenetic theory of cancerCancer is the result of multiple gene mutations Accumulation of mutations in different genes due to genetic or epigenetic variationUp to 1010 mutations over a human lifetimeGenomic instability Mutator phenotypeAneuploidyRearrangementTranslocationInversionDeletionSNP’sAmplificationClonalityTumors are comprised of clonal cell populations that all originate from a single founder cellDisregulated growth and then disregulated movementAre all cells dividing?Think that cancer stem cells are the only ones dividingProto-onco genesGenes that promote/ stimulate normal cell division and growthGain of function: by overexcitation or loss of regulation, proto-onco genes become onco genes, which stimulate hyperproliferationTumor suppressor genesGenes required for negative controlShut down cell division if activatedSo if you lose control via a Loss of function mutation, it leads to cancer1-2% of cancer is hereditary Ex: FAP (Familial adenomatous polyposis)Heritable cancer based on mutated copy (single) of APC gene on chromosome 5Keep growing-don’t stop divisionAPC=tumor suppressor geneRole in contact mediated growth inhibitionGet polyps in the SIDriver mutationsConfer growth advantage to cancer cellsCancer becomes worse with these mutationsPassenger mutationsOther mutations that happen in the course of cell division that do not confer growthEpigenetic variationDemethylation or acetylation of chromatin encompassing genes that stimulate cell division/migrationHypermethylation or histone deacetylation accompany genes that arrest cell division or mediate cell deathCell cycle controlAltered function of genes regulating the cell cycle can lead to dysregulation of cell division and excessive cell proliferationAbundance of different cyclins during the cell cycle that regulate transitions from one part to the nextMutate cyclinsGet cell division when cell shouldn’t be dividingApoptosisProgrammed cell deathBCL 2 level important (low for cell death)Cell death triggered by caspasesApoptotic bodies are engulfed by phagocytosisBax homodimer promotes apoptosisP53 induces BAX transcriptionInhibits BCL2 transcriptionThis stimulated cell deathP53 low function in cancerBAX transcription lowBCL2 highCell doesn’t dieRAS (protoonco gene)GF stimulates the cell proliferationsActivated when bound to GTPTells the cell to proliferateMutated and constantly active-cell always proliferatesP53 (tumor suppressor) DNA damage repairDNA damage promotes cell cycle arrest and fixing of the DNAMutation rate increases if p53 not workingRB1 (tumor suppressor)Inhibits TF’s when not phosphorylated Can be inherited- one copy damagedTriggers a cascade of genes that pushes the cell through the cell cycleNo longer binding E2F?Constantly pushes the cell through the cell cycleOnly one good allele neededMigration of metastatic cells away from the primary tumor siteEstablishes itself at secondary tumor siteGet blood vessels to oxygenate (angiogenesis)Metastatic cellsReduced expression of E-cadherin glycoprotein (reduced cell-cell adhesion)Increased expression of tissue metalloproteinases (TMP’s) (increase cell migration)Reduced interaction with tissue inhibitors of TMP’s (increase cell migration)LOF mutation in metastatic genes-leads to metastasisOr GOF mutationViral contributions to cancerMany people believe that cancer is caused by a virusOnco gene retrovirusesAcute transformation retrovirusesFirst IDed in chickens by Rous (RSV gene)RSV translated portions of a cellular gene that stimulates cell division (C-SRC)May pick up the gene from the genome while spreadingInserted into another genome and leads to overexpression (now 2 copies)Environmental contributionsRadiationSmokingOther factorsDrug designUse the exact path to develop drugsMany times specific to a certain mutationGleevecActs as ATP and binds the site that allows BCR-ABL to stimulate cell division when bound to ATPTrastuzumabBinds to HER-2 and induces its removal (down regulation)HER signals less intense and the cell therefore divides less often ................
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