Exam 4 Study Guide
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Date Created: 01/14/15
EXAM 1 REVIEW CHAPTER 1 amp2 0 Problems in replication of linear DNA strand gets shorter use telomers to elongate strand 0 Know chapter 2Sense vs antisense strand from chapter 1 know central dogma Supercoiling makes dna look difffernet generates topoisomers by doing that If delta k less than 0 negative supercoiling if more thanpositive supercoiling If 0 then no supercoiling Negative supercoiling more common bc provides less tension easier for strands to open up for replication and transcription 0 Diff between DNA AND RNA ribose ring and thymineuracil 0 Know whats major and minor groove and know how theyre diff in a b 2 form and how backbone phosphate arranged in 2 form 0 For double helix to be maintained need H bonds CHAPTER 9 Dna scondensation linked to scuffolds Focus on structure Euchromatin more loosely packed most transcription occurs in euchromatin o Dna element is centromere protein complex is kinetochore The spindles bind to the kinetochore Spndles come from MTOC there are 2 MTOC s in a dividing cell and they are on opposite sides 0 Key activitis of telomerase uses RNA template to make telomere longer Know overall general structure of telomere single stranded Telomere is on both laggingleading strand 0 Genetic maintianice centromere telomere kinetochore CHAPTER 10 Chromatin less compact hard to see under mic Histones make dna more contact while dna wraps around histones we get crossover supercoiling occurs 0 Lysine arginine cetoBonine know what can happen to them Insulator is a dna element 0 In general I ts naked its more active not wrapped around histonr CHAPTER 12 Replicon is the one unit that can replicate by itself Licnsing factors are the proteins that are available when needed 0 In bacteria oric will disappear after replication occurs In eukaryotes the ARS is there before and after 0 Know the binding protiens 6 of them abchu CHAPTER 13 Dna polymerases are key for elongation Know dna pol 3 and episilon and delta 0 Know enxymes in priming extension primer removal thing 0 EXAM 4 REVIEW CHAPTER 26 OPERON Modes of operon regulation 1 Negative control repressor protein prevents RNA polymerase from initiating transcription gene on by default Positive control activator required for RNA polymerase to initiate transcription gene off by default 3 lnduction transcription of an enzymecoding gene in response to appearance of its substrate 4 Repression transcriptional suppression by the product of its enzyme pathway lnducers inactivate repressorsactivates activator Corepressors inactivate activator or activate repressor Lac operon It is a negative inducible regulation gene is regulated by the substrate lac operon The lacl coding for lac repressor is always expressed at low levels The lac repressor binds to operator and inhibits RNA polymerase function Allolactose is an inducer that inactivates the lac repressor and also a substrate for Betagalactosidase IPTG lac operon Gratuitous inducer not substrate of induced enzymes lnducer binding causes conformational change in DNAbinding domain which prevents repressor from binding operator allostery Constitutive mutation mutation in operator that may either abolish expression or cause constitutive expression of operon Removal of lacl or inactivating mutations are denotes as lacl and these mutations cause constitutive expression of lac operon Accumulation of glucose inhibits transcription of lac operon catabolite repression Catabolite repressor protein CRP is required for transcription of genes it is a activator It is activated by cAMP inducer which activates the CRP which level is reduced by glucose high glucoselow cAMPinactive CRP The trp operon is negatively controlled and repressible by the level of its product tryptophan is a corepressor Tryptophan activates an inactive repressor trp repressor Trp Operon is also controlled by attenuation a special type of transcription termination Regulation of bacterial operons by controlling transcription termination at a site located before the rst structural gene Attenuator termination at which attenuation occurs Trp operon attenuation controlled by rate of translation of attenuator ORF depends on RNA secondary structure which is regulated by movement of ribosome lf trptRNA abundant ribosome moves quickly and secondary structure formed transcription terminates before structural 2 genes lf trptRNA low ribosome moves more slowly and secondary structure not formed structural genes are transcribed lf free trp level low and trptRNA level high transcription will start but terminate prematurely lf free trp level high transcription will not start regardless of trptRNA level CHAPTER 27 PHAGE o Lytic cycle Virulent phages follow this cycle 1 Viral DNA replication 2 Synthesis of structural proteins late genes 3 Progeny virus production 4 Cell lysis Lysogenic cycle Temperate phages follow this cycle 1 DNA integration ProphageProvirus phage genome integrated as a linear part of bacterial chromosome 2 No late gene expression 3 Bacteria are not killed 4 Induction when prophage is freed from lysogeny and phage enters the lytic cycle as a result of destruction of the lysogenic repressor it s cleaved triggered by adverse environment Lytic development happens by expression of phage genes in a speci c order divided into two periods Early period before DNA replication and Late period after onset of DNA replication Controlled by Cascade immediate earlyearlydelayed early middle late genes 0 Antiterminationresults in failure in termination and allows the same promoter to generate new transcript early gene expression continues because same promoter has to be used Lambda has 2 immediate early genes Both cycles have these 1 N antiterminator required by both lytic and lysogenic cycle to express the delayed early genes 2 Cro repressor Represses cl cro and N Both cycles 0 Three of the delayed early genes are regulators cll clll Q Lysogeny established when cl is expressed cll turns on repressor and clll maintains cll Both are required for cl expression The cl is lysogenic repressor The lambda repressor binds to CL and OR operators to prevent RNA polymerase from initaitng transcription of N and cro blocks lytic cycle Lambda repressor binding to OR stimulates its own gene cl autoregulatory circuit positive feedback The cll acts directly at the promoter PRE and clll protects cll from degradation Transcription from the promoter leads to synthesis of the lambda repressor and also blocks expression of cro Once lysogeny established CH and clll not necessary because of positive feedback of cl itself Lysogeny also required int expression integrase Int is expressed by Pl which is activated by CH The cll also promotes P antiQ which generates transcript complementary to Q mrNA Q expression is inhibited 0 p0 antiterminator required for late gene expression Iytic cycle only In presence of pN N product transcription continues to downstream of each gene lysogenic and Iytic Lytic cycle depends on antitermination by pN pN permits transcription from same promoters to continue past N and cro and allows transcription of delayed early and late genes Lysogeny 1 Immediate early N and cro are transcribed 2 Delayed early N antiterminates CH and clll are transcribed 3 Lysogenic establishment cll acts at the promoter cl is transcribed 4 Lysogenic maintainance Repressor binds to OL and OR cl is transcribed from other promoter Cro repressor which represses PRM cllambda repressor PR cro and PI N The lambda repressor and Cro can bind to any one of 6 operators with different af nitity When Cro binds to OR3 it prevents RNA polymerase from binding to PRM and blocks maintenance of cl expression inhibits cl gene Af nity for lambda repressor highest 01 Affinity for Cro highest 03 Lysogenic or lytic cycle depends on which repressor cro or cl occupies PrOr Critical event is whether cll causes sufficient synthesis of lambda repressor to overcome action of Cro Cll stability in uenced by clll and other host factors favorable environment for speci c cycle Basically cro will be transcribed the key determining factor is if cll causes sufficient synthesis of repressor to overcome this action and switch to lysogenic cycle CHAPTER 28 EUKARYOTIC TRANSCRIPTION REGULATION o Transcriptional regulation 1 Activation of gene structure on chromatin histone modi cation epigenetics 2 Initiation of transcription and elongation 0 Posttranscriptional regulation 1 Processing transcript 2 Transport from nucleus to cytoplasm 3 Translation of mRNA 4 Degradation of mRNA Activators positive regulation they work by interacting with basal transcription factors or with coactivators 3 True activators TFs binding basal apparatus at the promoter function on DNA or chromatin template Antirepressors chromatin remodeling proteins work only on chromatin template not DNA Some true activators are also antirepressors Architectural prtoeins Changes DNA structure bending Repressors negative regulation 1 Repressor protein that inhibits expression of a a gene typically by binding to an enhancer sometimes called a silencer DNAbinding and transcriptionactivation activities are carried by independent domains of an activator the DNAbinding domain DBD and activation domain DBD determines speci city for the target promoter or enhancer Types of DBDs 1 Zinc Finger DBD that typi es a class of TF that contain one more more zinc ions to help stabilize the prtotein Zing ngers from alpha helices that insert into major groove of DNA and Beta sheets that sandwich the zinc The Zinc ion is coordinated by cysteine or cysteinehistidine One protein has multiple Zn ngers Steroid receptor TFs that are activated by binding of a steroid hormone They have zinc nger domains for DNA binding but ZF is different from other ZFs SR are subfamily of ZF Helixturnhelix This is the DBD of lambda repressor and homeodomain hox proteins An arrangement of two alphahelices that form a site that binds to DNA Helix3 recognition helix inserts into major groove and Nterminal arms lies in minor groove HelixloophelixHLHThe region of the alpha helix that binds DNA contains basic amino acids residues Dimerization formed from 2 helical regions homodimer or heterodimer One is shorter alpha helix other is DNA recognition helix inserting into major groove Each helix connected through a loop Leucine Zipper Consist of stretch of amino acids with a leucine residue in every 7th position This stretch is hydrophobic A zipper in one polypeptide interacts with another to form a dimer Upper part short stretch of coiled coil structure where helices held together by hydrophobic interactions with the spaced leucine Lower part alpha helix interesting into major groove basic region binds to DNA bZlP o Chromatin remodeling Energy dependent by hydrolysis of ATP displacement or reorganization of nucleosomes during transcription Major role is to change organization of nucleosome at promoter resulting in activation or repression Remodeling complex does not have speci city for any particular target site on DNA It s recruited by activators or repressors that bind speci c DNA sequence Some of them can bind DNA in nucleosome Newly synthesized histones are acetylated at speci c sites in the cytoplasm and then deacetylated after incorporation into nucleosomes Histone acetylation usually associated with activation of gene expression Acetylation mediated by HATs Corepressors recruit the Deacylation by HDACs Absence of histone acetylation is feature of heterochromatin Bromodomain found in variety of proteins that interact with chromatin recognizes acetylated lysines on histones Chromodomain binds methylated lysines in histones o Methylation of DNA is associated with transcriptional repression Methylated histones are associated with transcriptional activation and repression depending on sites of methylation H3K4 ditrimethylationactivation methylated H3K9 or K27repression Methylation removed by KDM1 or jumonji family demethylases o H3K9 methylationtranscript Repression It is also linked to DNA methylation which alsotranscript Repression First H3K9 methylated by SETcontaining KMT then it recruits HP1 chromodomain protein which recruits DNA methyltransferases which methylate cytosine in CpG islands which reinforces histone methylation and deacetylations and represses transcription 0 During lnitiation activator or repressor binds to DNA chromatin or free DNA and recruits chromatin remodeling complexes andor HATHDACHMT Alternative histones H2AZ are incorporated to nucleosomes in transcribed regions 0 During Elongation Histone chaperonesFACT and Spt6 help disassemny and reassembly of nucleosome Immediate reassembly at back of RNA pol crucial to prevent cryptic transcription GAL genes galactose metabolism GAL 110 genes positively regulated by activator GAL4 functions as dimer There are 4 binding sites in UAS GAL80 bind GAL4 activated by GAL3and masks its activation domain GAL3 galactose inducible is activated and sequesters GAL80 in cytoplasm CHAPTER 29 EPIGENETIC EFFECTS Epigenetics study of heritable changes in gene activity without any change in the sequence of DNA epigenetic effect is due to a selfperpetuating structure that doesn t depend on DNA sequence 1 Covalent modi cation of DNA methylation 2 Chromatin Structure diff modi cations 3 A protein aggregate that controls the conformation of new subunits as they are synthesized ex prionproteinaceous infectious agent that behaves as an inheritable trait but doesn t contain any nucleic acid Active genes inserted within or near heterochromatin are inactivated Heterochromatin is nucleated at a speci c sequence nucleation and the inactive structure propagates along chromatin ber for a distance propagation due to epigenetic effect The closer a gene to heterochromatin the higher its chance to get inactivated nucleation and propagation Length of inactive region varies inactivation of genes in this vicinity causes position effect variegation PEV in drosophilaepigentic staus inherited by daughter cells Heterochromatin propagation may be inhibited by insulators active promoters and depletion of a protein factor Telomeric silencing analogous to PEV Genes translocated to a telomeric location show variable loss of activity Rapl binds telomere and recruits HP1 nucleation HP1 induces chromatin modi cation results in recruitment of more HP1 proteins propagation Another example is the mating switch lociMAT is active HML and HMR is inactive Inactivation of chromatin occurs by addition of proteins to nucleosome proteins bind to Nterminal histone tails that are subject to modi cation In Drosophila Suvar genes required for heterochromatin formation Evar genes inhibit it Heterochromatin formation at telomeres and silent matingtype loci in yeast involves a set of related genes silent information regulators SIR GENES Dosage compensation mechanisms employed to equalize the level of expression of Xlinked genes in the 2 sexes The entire chromosome is target for regulation Cisacting element called Xic Xinactivation center necessary and suf cient for X inactivation Only one copy of X is left active no matter how many copies of X chromosome present via counting mechanism Xist X inactive speci c transcript is required for X inactivation Xist regulated in negative manner by Tsix anti sense to Xist It forms double strand with Xist and further destabilizes Xist Accumulation of Xist on future inactive X leads to recruitement of Polycomp repressor complexes histone modi cations Heterochromatic state of inactive X is stable and Xist not needed for maintanenace of inactive state 5 of genes are still transcribed from the inactive X In Drosophila a ribonucleoprotein complex called MSL is found only in males and localized on X chromosome It contains 2 noncoding RNAs important for its localization to X and a HAT Transcription of genes throughout the male X chromosome is twofold increased In C elegans females use mechanisms to globally condense the 2 X chromosomes to reduce expression of each by half compared to male X chromosome CpG islandsmajor methylation site that is enriched in CpG dinucleotide 5 CG3 frequently found on promoter regions Repression can be achieved by 1 Methylation may inhibit activators from binding 2 Methylated CpG may recruit repressors MeCPl and MeCP2 De nova methyltransferases Dnmt3A and Dnmt3B methylate unmethylated CpG Hemimethlyated sites are converted to fully methylated sites by a maintenance methyltransferase Dnmt1 DNA methylation and heterochromatic state are mutually reinforcing DNA methylation promotes histone methylation and visa versa HP1 binds to H3K9me3 and HP1 binds to Dnmt1 which recruits the H3K9 methyltransferase 50 positive feedback These interactions contribute to stability of epigenetic states allowing heterochromatic region to be inherited Imprintingepigenetic changes during gametogenesis resulting in differential behavior genesalleles inherited from each parent caused by DNA methylation The imprinted genesalleles are inactive Imprinted genes are controlled by methylation of cis acting sites called DMDs differentially methylated domains or lCRs imprinting control regions Mouse chromosome H19 is epressed from maternal allele lgf2 from paternal allele When ICR is methylated the enhancer activates lgf2 When it s not methylated enhancer is blocked and H19 is expressed CHAPTER 30 REGULATORY RNA RNA functions as regulator by forming region of secondary structure intrainter molecular that changes the properties of a DNARNA Double strand formation can enhance or inhibit regulatory proteins from binding to target RNA Duplex formation may alter overall secondary structure of target RNA Reg RNA can regulate transcript and translation Riboswitch RNA whose activity is controlled by small moleculeligand Its an element in 5 UTR that can assume alternate base pairing con gurations secondary structure that can control its own translation or transcription attenuation Aptamer DNARNA region of ribozyme that binds to speci c target molecule Ligandbinding to aptamer in riboswitch can result in conformation change in RNA Riboswitch can be ribozyme Noncoding RNAs ncRNAs RNAs that don t contain open reading frames don t produce protein 1Antisense RNA RNA that has a complementary sequence to a target RNA quotNested Genesquot may produce antisense RNAs if transcribed from the opposite strand Nested gene gene that resides within another gene host gene Transcriptional interference Tl phenomenon in which transcription from 1 promoter interferes with that from another promoter transcription of one gene not product in uences transcription of another gene Occurs when nested genes are transcribed in opposite direction Cryptic unstable transcripts CUTs transcribed in opposite direction to target genes to make antisense rNA rapidly degraded by TRAMP complex recruits HDACs for gene inactivation effect is gene speci c doesn t extend to other genes Promoter Upstream Transcripts PROMPTs unstable 5 capped polyadenylated similar to CUTs Large intergenic noncoding RNAs lincRNAs originates from intergenic regions between genes Small RNAs sRNAs Bacterial RNA that regulates transcription translation and mrNA degradation stability Generated by small genes differ from miRNAs Most sRNAs work by base pairing imperfect with complementary target mrNAs antisense RNA which is aided by qu Protein CRISPR cluster of regularly interspaced short palindromic repreats RNAmediated dfense system of bacteria which protect it against viruses and plasmids CRISPR RNAs chNAS incorporated into effector complexes CASCADE and promote destruction of invading DNAs chNAs guide Cascade to target DNA and Cas proteins nuclease degrades it RNA lnteference RNAi Post transcriptional gene silencing PTGS process by which short antisense RNAs inhibit gene expression by translation inhibition or mRNA degradation of mRNAs complementary to miRNA or siRNA Choice determined by degree of base pairing between target mRNA and miRNA 2 higher degreemore likely mRNA degradation miRNA target sequences usually found in 3 UTR of mRNAs An mRNA can be targeted by mutltiple miRNAs 1 Short interfering RNA siRNA exogenous viral dsRNAs and arti cial dsRNAs and endogenous transposons and long dsRNAs microRNA miRNA endogenous short hairpin RNA transcripts Primary transcripts fold into a doublestranded hairprin structure with imperfect base pairing Pre miRNA processed by Drosha in nucleus premiRNAl processed by Dicer in cytoplasm miRNA loaded onto RISC which degrades passenger strand strand not used and delivers miRNA to target mRNAs 3piwiinteracting RNA piRNA endogenous long ssRNA Mainly function in nuceus to repress expression of transposons preserve genome integrity and control chromatic structure 2 CHAPTER 3 GENETIC ENGINEERING Cloning Vector DNA that can be used to propagate an incorporated DNA sequence in a host cell Contain selectable markers and replication origins to allow identi cation and maintenance of vector in the host Also have multiple cloning site MCS which contains various restriction enzyme recognition sequences 1 Plasmidextrachromasomal circular DNA molecule that autonomously replicates inside bacterial cell random segregation Phage derivatives of bacteriophage lambda linear DNA molecules whose region can be replaced with foreign DNA without disrupting its life cycle only vectors with insert may be packaged used for selection Cosmids extrachromsomal circular DNA combinds feautures of plasmids and phage larger insert can be used Bacterial Arti cial Chromosomes BACbased on bacterial miniF plasmid Yeast Arti cial Chromosomes YAC contains telomeres origin of rep yeast centromere and selectable marker for identi cation in yeast BlueWhite screeningalpha complementation allows identi cation of bacteria that contain vector plasmids with an insert LacZ gene has deletion at 5 end alpha fragment is lacking and only w fragment is expressed Alpha fragment 3 4 5 provided by cloning vector Insertion of DNA fragment at MCS will disrupt expression of alpha gragment Vector with insert white colonies vector without insertbue clonies Expression vectors contains promoters that allow transcription of any cloned gene Include promoter poly A signalterminator and other elements Reporter genes used to measure promoter activitiy in vitro and invivo desired to be quanti able DNA Delivery methods 1 Intro of DNA into bacteria and yeast transformation transduction and electroporation In eukaryotic cells transduction viral vector transfection chemical method liposome electroporation microinjection gene gun Nucleic Acid Detection nucleic acids absorb light with wavelength of 260 nm absorbance is proportional to concentration of nucleic acids 1 Southern blot Involves transfer of DNA from a gel to a membrane followed by detection of speci c sequences by hybridization with a labeled probe Transfer can be achieved by physical pressure or electric current 2 Northern blot detection of speci c RNA like SB 3 Polymerase Chain Reaction PCR ampli cation of speci c DNA sequence Depends on the use of thermostable DNA polymerases that can withstand multiple cycles of template denaturation Necessary components are DNA template therm DNA polymerase 5 and 3 primers short ssDNA with 3 OH dNTPs and Mg2 Amplicon is the primertoprimer dsDNA products of PCR or RTPCR Reverse transcription PCR RTPCR ampli cation of speci c RNA sequence Uses reverse transcriptase to convert RNA to DNA followed by a PCR reaction DNA copy from RNA cDNA Hybridization base pairing of labeled nucleic acid probe to identify speci c nucleic acids by complementary sequences 6Autoradiography Method of capturing image of radioactive materials on lm Fluorescent in situ hybridization FISH utilizes uorescencelabeled probes that bind to speci c DNA sequence within chromosome Classing staining doesn t detect small changes translocation deletions etc It identi es chromosomal aberrations and locating enes on chromosome and chromatin Chromosome Paintinguses 2 4 5 7 multiple uorescent probes binding all along a particular chromosome visualize one chromosome Spectral karyotyping SKY visualize all pairs of chromosomes labeled with different assortment of colors DNA separation techniques 1Gel electrophoresis separates linear DNA fragments by size and shape using electric current to cause DNA to migrate toward a positive charge Migration rate is inversely proportional to size of DNA fragment Smaller size travels faster Supercoiled DNA migrates faster than relaxed Supercoiledgtlineargtrelaxed DNA sequencing chain termination sequencing uses dideoxynucleotieds ddNTPs to terminate DNA synthesis at particular nucleotides They are uorescent tagged and gel electrophoresis allows automated DNA sequencing Next generation of technique are higher less time and cost effective Western Blotting separation of proteins on an SDS gel transfer to membrane and detection using antibodies DNA Microarrays these comprise known DNA sequences probes spotted or synthesized on a small chip lt s opposite to blotting methods where sample are immobilized 1Gene expression pro ling performed using labeled cDNA from experimental samples hybridized to a microarray containing sequences from all ORFs of organism being used 2 Single Nucleotide Polymorphisms SNP these arrays permit genomewide genotyping SNPs are DNA sequence variation in a single residue 3Array comparative genome hybridization array CGH allows detection of copy number changes in any DNA changes compared between 2 samples array chiptiling array Chromatin immunoprecipitation ChlP allows detection of speci c proteinDNA interactions in vivo ChlPonchip ChlP chip integration of ChlP and microarray Used to investigate interaction of proteins and DNA in vivo on genomewide basis ChlPseq can be used to obtain similar info Transgenics organisms created by introducing foreign DNA transgene into germline Expression levels of transgene vary depending on transgene copy number and site of integration Gene knockouts gene deletions Gene knockin replacement of a gene with an alt version of same gene or inserting new gene into speci c locus Gene knockdown reduction of gene expression Conditional knockoutknockinknock level of expression controlled Crelox system used to make these Two loxP sites introduced to anking regions of exons Deletion of oxed exonsframeshift mutation premature stop codon Deletion of oxed alleles achieved by mating mouse with a transgenic mouse expressing Cre recombinase Cre expression can be temporally andor spatially regulated How to make knockoutknock in mice 1Construction of targeting vector DNA seq with desired modi cation anked by DNA seq identical to those in target genomic locus 2Transfecting ES embryonic stem cells with targeting vector More frequent with random integration than homologous recombination 3Screen ES cells with HR occurrence of successful HR can be detected by using 2 selectable markers one incorporated with integrated gene positive selection and other which is lost when recombination occurs negative selection 4 ES cells with desired modi cation injected into blastocysts Engineered ES cells will contribute to development of mice When mutated ES cells contribute to germline next generation of mice may be derived from modi ed ES cell YeastTwo Hybrid Screening ldenti y proteins that interact with protein of interest Protein of interest bait is fused to a DNAbinding domain of transcription factor cDNA library prey is fused with activation domain of TF Promoter for a reporter gene contains DNA seq that the DNAbinding domain binds to and reporter gene expression require for growth of yeast Interaction of bait and prey reconstitutes an active TF and enable yeast growth
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