BSCI222/DR.KOCHER: Test 3 Review Sheet
BSCI222/DR.KOCHER: Test 3 Review Sheet BSCI222
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This 12 page Study Guide was uploaded by HannahJ on Saturday November 28, 2015. The Study Guide belongs to BSCI222 at University of Maryland taught by Dr. Kocher in Fall 2015. Since its upload, it has received 161 views. For similar materials see Genetics in Biological Sciences at University of Maryland.
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Date Created: 11/28/15
Chapter16 ● as gene expression increases,energetic costs increaseas well as regulatory speed ● bacterial operonincludesa singlepromoter (directs transcription),andupstreampromoter, and a Shine-Dalgarno sequence ● repressors are proteinsthatbindto operators andprevent transcription of structural genes through negativeinducible control ● inducers bind to repressor andcauses it to beunable to find to operator through anallosteric (shape) change ● thereareinactive regulatorsthat can’t bindto operators ● corepressors bind toregulators andcause them tobecomeactiveand prevent transcription through positiveinduciblecontrol ● lac operonconverts lactose to glucose ○ 3genes ○ without lactose: regulator proteinbinds to operatorandinhibits transcription ○ withlactose: allolactose binds to regulator and it becomesinactive ○ Ocmutations:operator, prevents binding siteforrepressor ○ multiplerepressorbind sites, binds asa tetramer ○ I= repressor. P =promoter.O = operator.Z =beta galactosidase.Y =permease ● partial diploidshavetwo copies of lac gene(one from F plasmidand one chromosomal copy) ● I(s) is asuperrepressor thatisalways boundto the operator evenin thepresenceofallolactose ● catabolite activator proteinis a secondmechanismofcontrol (CAP) ○ when high levels of cAMP bindtoCAP, itthen binds to the DNA, whichhas an activating effect ○ cAMP levels reflect energylevel ofthecell ○ low transcription without CAP, induces bendthat promotes transcription ● TRP operon ○ works under negative repression ○ repressoris normallyinactive, so transcription takes place ○ when there arehigh levels oftryptophan,it binds to repressor,allows it to bind to operator and stop transcription ● attenuation of transcription is asecondmechanism forcontrol ○ secondary structure of 5’UTR, 2TRPcodons next to each other ○ when TRP levels arehigh,regions 1 and2 pair, and3 and4 pair creating a terminator loop next to astring of U’s, stops transcription ○ when TRP levels arelow,regions2 and3pair andno hairpinforms, allows transcriptiontocontinue ● ompFisoutermembrane porinthat is a non specifictransporter for osmoregulation ○ when osmolarity islow, lotsofompF aremadeand placed in membrane ○ when osmolarity ishigh, micF gene producedmicFRNA that binds with 5’end of ompFRNA and blocks ribosomebindingsite to stopprotein production ● riboswitch offiswhena small regulatorymolecule binds to a riboswitch(ona mRNA) and stabilizes a secondary structure ○ creates aribosome bindingsite that is hidden andtranslocation can’ttakeplace ● riboswitch onhappens withouta small molecule, adifferent secondary structureis produced and translocation takes place because the binding site is exposed ○ transcriptionaland translationalcontrol ● ribozymeis a secondary structure andsmall molecule interactions ○ creates and enzymeforcleavage Chapter17 ● potentialregulation points in eukaryotes include ○ chromatin structure, initiation oftranscription, processinganddegradation, mRNA localization, translocation, proteinprocessing/localizationand phosphorylation ● chromatin structure can bemodified by pushingnucleotides asideof promoter ○ use chromatin mapping by taking asample, cutting with nuclease,andsequencing the extracted DNA ● nucleosome deficientregions are spacedright next to anucleosome, andthen regularlyspaced through therest (low points of models) ● chromatin remodeling complexes areproteins that bind to particularDNAsites andreposition nucleosomes ○ allowstranscriptionfactors to access promoters ● histonemodificationsalter chromatinstructureandallowtranscription factorsto access DNA through either methylation (nucleosome remodeling factor NURF, replaceH with CH3)or acetylation (losecharge interaction andweakenbonds) ● DNA methylation also alters chromatinstructure ● CpG islands are areas withGC orCGsequences ○ 60-90% of mammalCpGs are methylated ○ clusters foundnear transcriptionstart sites ○ methylation of Crepresses transcription by directly blocking transcription factors ● siRNA creates induced transcriptional silencing bybinding to proteins in RNAinduced transcriptionalsilencing complexwhichthenrecruits a methylating enzyme ● PIC is the pre-initiation complexwhich is very complicatedandis different at every promoter ● promotertrap**** ● response elements coordinateexpressionfroma commonstimulus ○ various proteinsmay bindtoupstream responseelementstostimulate transcription ● insulators areboundary elements thatlimit interactions of enhancers withother genes that limitthe spread of changesin chromatin structure ● CCCTC-binding factor loopstheDNA and blocks the target ● polymerase pausing hasbeenlearned about through the assembly oftranscriptional complexes of yeast ○ proceeds without pausing inyeast ○ evidenceofregulated pausing ofpoly II inmetazoans ● chIP seqis achromatinimmunoprecipitation ○ bind protein is cross linkedwith formaldehydeto isolatechromatinthrough the use of specific antibodies which reversecrosslinking.usePCR to amplify ● processing and degradation are post transcriptional regulators ○ alternative splicingcontrolled by asplicingfactor (sex determinationinsfruit flies through SXL sex lethal gene) ● mRNA degradation happensthrough3 different pathways ○ removingthe5’ cap leads to5’--->3’ degradation ○ 3’--->5’ leads to degradationof polyA tail ○ endonucleasecleavage atinternalsites ● siRNA can beused to degradespecificmRNAvia RISC ● RNA bindingproteins canrecognizesequences in3’ UTRs andattach to microtubules for transport through cell (mRNA localization) ● translation is a formofposttranscriptionalregulation (T Cells) through the regulationof initiation, which increases initiationfactors when needed ● attenuation impacts translation ○ PUF and argonaute proteins bindto3’ UTR oftarget geneand inhibit activity ● miRNA are diced and then can bindtomRNAto inhibittranslation ● proinsulin protein isprocessed to produceactive insulin ● phosphorylationis a post translational regulator ○ add phosphateto aminoacid ofprotein, which can switchonor off ○ kinase: add,phosphatase: remove ○ can induce a conformational change ○ usually occurs onserine, threonine, andtyrosine ● p53 isa tumor suppressorthatregulates the cell cycle Chapter18 ● somatic mutations occur innon reproductive cells and arepassed alongthrough mitosis ● germlinemutations occur in reproductivecells and are passed through meiosis and sexual reproduction ● can usepigmentation mutations or newly auxotrophic bacterial cells to assayfornew mutations and find mutationrates ● microorganisms havea rate wheremutants/cell equals mutations/replication ● multicellularorganismshavearound1 x 10^5phenotypicmutationsper gene ● universally, each gamete hasaround 1-2 newmutations ● alpharadiation: can be stoppedby paper,helium ● beta radiation: needprotectionfrom, electrons ● gammaradiation: need protectionfrom, energetic photons ● neutron radiation:free neutrons ● beta and gamma undergo ionization whichistheprocessofknocking electrons loosefromtheir orbitals ● radiation can cause single or double strandedbreaks and canchange bases ● mutational rate isproportional to radiation dose ○ need abig increase to double rateof mutation, 10%more radiation can increase frequencyof raredisordersby 1% ● the Ames Test is usedto assayfor mutagenicityofcompounds ○ vile of bacteria, rat liverextract, potential mutagen,plateitinhistidineand count the numberof revertants, uses prokaryotes ● HERP index standsfor human exposure:rat potency, tells therelative risk of mutagens ● genotype mutationsinclude base substitutions, insertions, deletions, transitions (A/GorC/T, changeto sametype ofbase) andtransversions (Aor G/C orT, different typeofbase) ● phenotype mutations include ○ missense: code for different aminoacid ○ nonsense: newcodonis a stop codon ○ silent: new sequence encodesfor same amino acid ○ forward: wild type to mutant ○ backward: mutant backto wildtype ○ suppressor: ■ intragenic: secondmutationin the same generestoresshape/function ■ intergenic: suppressortRNa, rescues stopcodon ● errors occur through problems inreplication, spontaneousdamageto baes,or chemically induceddamage ● tautomers are mis pairingsand arenot common ○ T and G wobble pairing, CandA wobblepairing ○ are oftenfixed due to distortionoftheDNAstructure ● slippagecreatesinsertionsand deletions ○ when the new strandloops out extra nucleotidesadded ○ when the template strand loops out youlose nucleotides ● triplet expansion is agenetic disorder wherea trinucleotiderepeat expands ● spontaneousdepurinationis when youlose abasein a normalchain to create an abasicsite ○ can lead to mutations ifunpaired becauseanA is usually addedwhen thereis no template ● deamination is when a cytosine changes to auracilthrough the loss ofan NH2group ○ easy tofix ○ methylation of CpGthendeaminatebecomesathymine ● thymineand5-bromouracilbindinthe samespots but 5hasa higher ionizationrate andbinds withguanineover adenine ● intercalating mutagensstack in betweenbases (largegapin helix) and usedto visualizeDNA in gelelectrophoresis ● thyminedimers are made by UV light andarewhen adjacent T’s covalently bondtogether ● thereisan enzyme that finds basesthat aren’t properly paired, usemethylationto detect andfix ● suicide enzymes cause degradation,need toberemadeaftereachuse ● baseexcisionis frequent, location andremoval of damaged basesto produce an AP site ● excisionnucleotides work on bigger sections thatbaseexcisions,removebulkyadducts Chapter19 ● restriction enzymes protect against viruses bycutting at palindrome sequences ○ staggered cutsproduce sticky ends ○ straight cutsproduce bluntends ● recombinant DNA (combingdifferent sources of DNA) is made by cuttingwith thesame moleculeto make complementary sticky ends andpairing themtogether ● gelelectrophoresis is usedto separate DNA fragments by size (intercalatingdyes) ○ place DNA inwellson negative pole,sendcurrent,DNAmoves to positive end, smaller fragmentsmove quicker and willbefurthest fromwells ● southernblot isusedtomake the gel electrophoresisstable andpermanent ○ denature the doublestranded DNA,put membraneontop of gel,buffer drawninto top layerof blottingpaper andcarriesDNA with it ○ transfer to nylon membranethat is positivelycharged ● RFLP’sare restrictionfragment length polymorphisms that are used tohelp mad human genomes ● simplecloning vectors need 3 things: ○ origin of replication, restriction sites(s), selectablemarker ○ plasmid andforeignDNA cut atthesame site,stickyends annealthetwotogether ○ problems: few restriction sites, hardto distinguishrecombinant from non ● more useful cloning vector has multiple cloning sites insidelacZ and usesblue/white color screening ○ foreignDNA inserted into lacZ, lacZ- transformed byplasmids ○ blue ppt: make B-galactosidase by cleavingX-gal NONRECOMBINANT ○ whiteppt: don’t makeB-galactosidase,can’t cleaveX-gal RECOMBINANT ○ bacteria with no plasmid won’tgrow ● fancy expressionvector is a operon sequence that allows insertedDNA to transcribe/translate ○ regulate desired gene ● oligonucleotides canbe created tohit onetarget sequences,pairandact asprimerforDNA synthesis, creates a molecule witha single mismatchedpair, onesingle nuc. changed ● PCR isa polymerasechain reactionforDNA amplification ○ heat to separatestrands, cool quicklytoallowsingle strand primers to anneal, heat again to start DNA synthesis ○ produces strands between primers ○ 2^(number of cycles)=# ofshort strands ○ taq polymerase stable at95degrees ● microsatellite isa sequence that is repeatedmanytimes inDNA,individualistic ● Ti plasmids are tumor inducing by invadingatwound andtransferring partofplasmin into chromosomesto addforeign DNA into plasmid and it is thentransferred to plant cell ● bacillus thuringiensisisa naturally occurring bacteriumthatistoxic toinsects wheningested, used as repellent, Bt gene into plants ○ delivers sufficient dosage, only those whoeatit die ○ impacts natural selection, how does it impacthumans? Chapter20 ● cystic fibrosis gene is autosomalrecessive,1 out of 3000,mucus in lungs ● human genome is 36Gbp ● each chromosomehas 130Mbp ● 1 cM is 1 Mpb(hardto map beyondthis stage, thenneed to makeclones to sequence) ● there are 8 genes perMbp ● positional cloning steps include:mapping,chromosome walking,sequencing,functional analysis ● RFLPscan beused to see differencesbetween 2genomesequences throughan association study (compare frag. sizeswith normal andinfectedphenotypes) ○ found association between chromosome 7 markersandcystic fibrosis ● partial digestion isthe first stepin making aDNAlibrary ○ digest DNA with restriction enzymefor limitedamountsoftimes (onlysome sites cut ineach molecule) tocreateoverlappingfragments ○ join each to cloning vector andput intobacteriato create set of clones some havegene of interest ● screening libraries is the second step ○ use radioactively labeled probes and stick anitrocellulose disc ontop ofbacterial colonies (somewill stick) ○ denature DNA, labeledprobehybridizes with complementaryDNA,detects presence of gene ● chromosomewalking is when aprobethat is complementary to the end of clone A issued to find overlapping clone B, etc ○ can’t tellif you are ‘walking’ awayor towards geneofinterest ● chromosomejumping usespartially digested DNA that areligatedtogether at ends to make long fragments ○ the circular molecules are cutby restriction enzymesto makesmaller fragments (some have beginnings and ends oflonger fragments next to each other) ○ complementaryprobes usedat end ofonecloe to finddistinct clones ● cystic fibrosis gene is around 250000bplongs with 24exons andencodes for1480amino acid chlorinechannels ● ddNTP can have a fluorescent labelto automate DNAsequencing ● public genomeprojectstrategy: ○ detailed genetic linkage maps, physical maps, assemble ● fingerprinting clones are made by cuttinglargepieces ofDNA withdifferent band sequences withrestrictionenzymes andoverlapping clones makecontigs(physical map ofgenome) ● minimum tiling isthe processof choosing clones that overlap the least so you don’tsequence more than youneed to ● privategenome project strategy: ○ sequence expressedgenes through mRNA to makea cDNAlibrarywithout physical maps ● cDNA library construction: ○ short oligo(dT) chains linkedto cellulose incolumn, pass RNAthrough and poly A tailspair with chains so mRNA kept incolum ○ oligo(dT) primers annealto polyA tailto providea 3’OH group,reverse transcriptase synthesizes ○ RNA DNA hybrid treatedto partially digest RNA, DNA polymakes a second strand usingtheRNA asprimers ● wholegenome shotgun sequencing has todo withrepetitiveDNA, don't’ know where toplace them ● paired end reads consisted of 3libraries of different insert sizes (4/10/40kb),scanned repetitive sequencesof diff sizesand sequence both ends of clone, softwareto place pairedreads at appropriate distancesfromeach other ● post project,sequence determinedas DNAis synthesized, not after ● Illuminasequencing: ○ microscope slide with 8channels, DNAtreated with ligateadaptorsonbothends, put inside cell channels ○ bridgeamplification by adding unlabelednucleotides, adapters link,replicate ○ DNA becomes doublestranded, denatured, amplify,add tagged nucleotides, make withnew strand and take pics after eachnucisadded ● Pacific Biosciences: ○ no amplification needed,single moleculedetection, DNA synthesizedinreal time ○ polymerase inbottom ofzeromodewaveguide, smallamountoflight can come through to see whichnucis associated through acolorchange Chapter21 ● can getnew phenotypes throughmutations, or epigenetics (DNA andhistonemodifications, noncoding RNA) ● epigenetics isa change inthephenotypethat is potentially heritable without changingthe DNA base sequences throughmethylation of DNA,histonemodification, small RNA interactions ● methylation can switchgenes on and off,cytosines ● histoneacetylationundoes the normaltight packingof nucleosomes to createloosepackaging, new genes are exposed to transcriptionfactors andthusexpressed ● small RNA through the direct andindirect recruitment model ● honeybees use demethylation to distinguishbetween worker and queenbees ○ demethylationleadsto re-activation ofFASlocus expression byhistonedeacetylase inhibitor, opens chromatinandactivates gene expression ● mice use methylation for fur color ○ increase methylation inpseudoagoutimice ● epigeneticmodifications can be passedthrough generations that canbeimpacted bynutrition, environment, famineetc ○ vinclozolinis afungicide that is an endocrine disruptor that impacts F1/2/3 generations ○ early lifestress in miceleads to alteredmethylation in brain ● X-inactivation: ○ X-inactive specifictranscript and repressor RNA expressedononeX ○ RepA bindstoPRC2 onfutureinactivated X, Xist RNAspreads,increasesPRC2 concentrationwhich engages weaksites onXchromosomes ○ competitionbetweensites determines which chromosomeis inactivated ● paternally imprinted iswhenexpression of the father's allele isloverbyepigenetic modifications ● as development of fetus takes place, methylation is removed, increases at post implantation (needstobe removed to have fetus growproperly) ● paramutationis achangeingeneexpressionon onelocus inducedbyallelicinteractionsat a different locus ● odortraining in mice: ○ pumpin badsmell and shock feet, learn connection, neurons aremore activewhen smell is sensed ○ offspring are nervous with loud sound smellis there Chapter22 ● fruit fly egg develops into hollowcylinder ofcells, segmentationoccurs after a fewhours ● 3stages beforepupa,metamorphosis ● sperm and eggfuse to make single celled diploid zygote, nucleardivisionsto createsingle multinucleated cell (syncytium,nuclei move to edges anddivideto create syncytial blastoderm, cellmembranesform aroundeach nucleus creatinga layer ofcells surroundingembryo(cellular blastoderm( ● 2 hour embryo: anterior posteriorand dorsal ventral axisestablished ● 10 hour embryo: # andorientationof body segments established ● embryonic patterning of adult structures is storedinthe imaginal discs ofthelarvae ● dorsal-ventral istopto bottom ○ embryonicmembrane with Toll abovevitelline membrane, ventral follicle cellsattach to bottom where sulfated pipetarget placed, activateseasterprotease and interactswith Toll ○ maternal localizationof pipe sulfotransferase, easter serineprotease,localdistribution of spaetzle binds to Toll, signal that allows dorsal enternucleus and change transcription ● anterior-posterioris front to back ○ bicoid mRNA localized atanterior end, forms gradient withhighconcentrationat anteriorend, involvedindevelopmentofanterior structures ○ nanosmRNA localizedat posterior end,after fertilization nanosmRNAmakes nanos protein, concentration andinhibits formation of anteriorstructures ○ internal processes to keepgradients from equaling out ● gap genes deleteadjacentsegments(eliminateanterior segment) ● pair rule deletessome part of a pattern ineyer othersegment (delete even number segments) ● segmentpolarity effects polarity of segments, part is replacedbymirror image of part of another segment (posteriorhalf replaced withmirror imageofanterior half) ● promoters comput position of bicoid andnanos alonggradient,act basedonwhat is aroundto help andor blockbinding ● mutant examples: ○ bithorax: wings instead of halters ○ antennapedia: legs insteadofantenna ○ proboscipedia:legsinstead ofmouthand antennae ● hox genescontrol vertebrate segment identity and are similarbetweenspecies andthose inthe sameorder ● mammals have 4 clusters of hox genes whosearrangement correspondsto sequence in which genes areexpressed along anterior posterior axis ● hox proteins aretranscription factorsthat regulategeneexpression ineach segment ● ABC Model for floraldevelopment ○ class A genes produce sepalsin 1st whorl ○ class A and Bgenes producepetals in 2nd whorl ○ class Band C genes produce stamens in 3rdwhorl ○ class Cgenes produce carpels in 4th whorl ● early experiments showthat somatic tissues havecompleteset of geneticmaterial and differences in expressionduring development arenot dueto gene loss ● early embryoniccellsarepluripotent, can grow into anytissueand can reversetostemcells ● genes rearrangeinthe development ofantibodies ● lymphocytes comefrom stemcells inbone marrow ● B cells madein bone marrow, T cells madein mature thymus ● each B-lymphocyte isspecific for 1antigen, whenit binds to B cell,the B celldivides which is the primary immune response ○ some become antibody secreting plasmacells,somebecomememorycells (second immune response) ● immunoglobulin molecule has4chains with anantigenbinding sitewith variableregions, joining regions,heavy/light chainsanda constant region ● antibodies are differentbecause ofsomatic recombination, NOTsplicingbecause it isnot RNA ● 10 millionpossible Bcells ● Tcells have alpha and beta chains and work in thesame way to create1 million different shapes Chapter23 ● cancerisan abnormal proliferationofcells responsible for 1 in5 USdeaths, competes with normal cells for space and nutrients ● carcinoma begins inthe skin or in tissues that line internal organs(epithelial) ● sarcoma begins in the bone, cartilage, fat, muscle, orblood vessels ● blastomas begininstem cells ● leukemiabeginin blood forming tissue (bone marrow) and increasesthenumberofabnormal blood cellsintheblood ● lymphoma andmyeloma begin intheimmunesystem ● centralnervous systemcancers beginin the brain and spinal cordtissues ● cancerrates are impacted byenvironmental andsomaticmutations ● Alfred Knudsonfound that single cellsrarely undergo2somaticmutationsthat result ina a single tumorin 1 eye ○ apredisposed person inherits one mutation, undergo anotherto create caner ● multiplemutations requiredto producecancerous cells ● tumorsbecome dominantby mutants that competewell ● a cell that ispredisposedto proliferateata highrate, the2ndmutation causes rapidcell division, 3rdmutation causes structural change,4th causes it to divide and invadetissues ● genes cancause cancerby encouraging cellgrowthand division throughmutations in proto-oncogenes andby removing controls ongrowthanddivision through mutations in tumor suppressors ● proto-oncogenes usually produce factors that stimulatecelldivision, mutant alleles tend to be dominant ● tumor suppressors normallyproducefactors thatinhibit celldivision,mutant allelestendtobe recessive ● cellcycle regulators ○ MPF: mitosis promotingfactor ○ CDK: cyclin-dependent kinase ○ when retinoblastoma proteinandE2F aretogether it is inactive, cyclin andCDKhigh, RB becomes phosphorylatedit separates for E2F,whichthenbinds to DNAand stimulates transcription ofgenes ○ p53 isa tetramer transcriptional activator, that integratessignals ofDNA damage ○ p21 is produced whenp53 is made,helps to fix damagedDNA ● signaltransduction pathway ○ bindingof growth factorto receptor leads to conformationalchange which allowsan adaptor molecule to bind and link to Ras,which binds to GTP ○ causes asignal transduction cascade, which leads to activationof transcription factors promoting growth ○ mutations of Ras stimulatesignal transduction pathway,stuck inonposition ● defects inrepairand errorsin replication cancause cancer ○ 90% hereditary cancers due to mutationsin components ofmismatchrepair ● telomeraseisnormallyregulatedin somatic cellsto limit the numberofcell replicationcycles ● telomeraseisupregulated in tumorcells, allowingunlimitedcycles(passHayflick limit) ● HeLa cellsare an immortal cell line used for research ● overexpressionof angiogenesis promotingfactorscontributes totumor growth (allows the tumor to better use oxygen and nutrients throughthevascular system) ● metastasis is the abilityof a tumortomovearound and invade other tissues ● palladinmutationdisrupts actincytoskeletal network ofmesenchymal cells, leads toincreased mobility ● dicer processed miRNA regulatesexpressionpost transcription through RISC ○ lower expression of let-7miRNAleads to overexpressionofRas ● deletions, inversions,translocations impact geneexpression ● NIH Cancer GenomeAnatomy Project wanted tocharacterize geneexpression atvarious stagestoimprove detection, diagnosis, and treatment ● colon canceristhe lossof anormal tumorsuppressor gene APC,creates a benign, precancerous polyp, Rasactivationleads toadenoma grows, lose p53 tocreate a carcinoma malignant tumor ● retrovirus inserts RNA through reverse transcriptionandinsert hosts chromosome nextto proto-oncogene which incorporatesinto virus, gets mutated and is inserted back into host ○ mutation and rearrangement of host genes in viral intermediate ● HPV is started in epithelial cells, insertions canchangegene expression ○ gardasil vaccine:recombinant virus like particles lackviral DNAthat can’t makecancer but trigger antibody responseto protectinfuture ● transmissional tumor intasmaniandevils throughbites ○ transfer cellsand all areso closelyrelated basedon immune system so the cells don’tdie
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