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Date Created: 04/24/15
362014 I Basic Molecular Genetic Mechanisms O O O O Transcription takes info encoded in base sequence of DNA create preRNA RNA processed introns taken out poly A cap and so on leaves nucleus Translation into protein Nucleotide Adenosine monophosphate AMP base attached to ribose at 1 Carbon I Difference between ribose and deoxyribose deoxyribose has H on 2 while Ribose has OH I DNA synthesized 5 to 3 direction I DNA Major groove and minor groove 0 Allows proteins and RNA to bind to DNA 0 Double helix is exible 0 Protein interaction can bend DNA 0 Bending of DNA is critical to dense packing of DNA in chromatin Why did DNA evolve to be carrier of genetic info in cells as opposed to RNA I Hydrogen at the 2 position in the deoxyribose of DNA makes it far more stable than RNA which has a hydroxyl group at the 2 position During replication and transcription of DNA the strands of the double helix must separate to allow the internal edges of the bases to pair with the bases of nucleotides being polymerized into new polynucleotide chains I Easier to spate between AT because 2 H bonds instead of 3 in GC 0 Higher percentage of GC the higher the melting temperature of DNA 0 Good info for PCR Three dimensional geometry of DNA although eukaryotic nuclear DNA is linear long loops of DNA are fixed in place within chromosome torsional stress and consequent formation of supercoils also could occur during replication of nuclear DNA I Topoisomerase nick strand of DNAlt allow to unwind and relieve tension ligases reattach strands RNA conformations due to base pairing in on self I Secondary structure 0 Hairpin 0 Stem loop I Tertiary structure I Pseudoknot two stemloop RNA synthesis I Also 5 to 3 I Phosphodiester bond I RNA polymerase recognize where to bind unwind helix read template strand assemble complementary strand progress across strand recognize when message is over I Bacteria end of RNA polymerase GC hairpin I Prokaryotes vs Eukaryotes I P transcribe and translate at same time uninterrupted operons promoters and operators no post transcriptional processing general polymerase and multiple sigma factors gene clustering common transcribed from a single RNA operons single promoter for gene clusters 0 Transcription initiation promoter sequence positions polymerase sigma factors recognize promoters I E no transcriptiontranslation coupling intronsexons promoters and enhancers splicing and capping and polyA tailing polymerases 111 and III almost no gene clustering each gene has own mRNA each gene has own promoter 0 P01 I most rRNA genes 0 Pol II protein coding genes miRNA genes plus genes for some small RNA s 0 Pol III tRNA genes SS ara gene gens for many other small RNAs 0 Transcription initiation in Eukaryotes I Transcription factors bind promoter s TATA box I Binding of additional general TF s I TF s positions polymerase I RNA polymerase tail becomes phosphorylated I TF s dissociate and polymerase initiates RNA synthesis 0 Eukaryotic RNA are processed before leaving the nucleus I 5 end capping methyl guanine cap I 3 end poly A tail I RNA pol Tail has capping factors adds cap and poly A tail to end I Splicing I Removes internal noncoding sequence introns from coding sequences exons I Mature mRNA exons only Will be exported to cytoplasm I Over 100000 proteins around 20000 genes alternative exon splicing O Fibronectin produced by fibroblasts contains exons EIIIA and EIIB alternative splicing of the fibronectin primary transcript Fibroblast has EIIB and EIIIA to bind to ECM surface of cell Hepatocyte do not splice these soluble not bound down an help form clots O Decoding an mRNA into protein I Translation mRNA lprotein I mRNA AUGC three nucleotide combinations 64 combinations I 20 amino acids I match three nucleotide combination to amino acid I three binding sites in RNA cognate tRNA comes in and binds A site tRNA in A site moved to P site tRNA in P site moves to E site Where it is ejected I redundancy in genetic code codons to Amino Acids I 1 tRNA per amino acid I tRNA has wobble effect I multiple Reading frames I depends on Which base starts at if shift by one or two completely different amino acid sequence I translational machinery I ribosomes I aminoacyltRNA aatRNA synthetase I mRNA I initiation factor 3182014 I elongation factors I release factors high energy ester bond between tRNA and amino acid I proof reading function in aminoacyltRNA synthetase ensure wrong amino acid isn t put in aa binds to 3 end of tRNA I anticodon loop has anticodon where binds to codon within ribosome Ribosome decodes mRNA message I Ribosome ribosomal RNA 0 Ribosomal proteins 0 Prok 7OS SOS 308 O Euk SOS 608 408 O S density sphedberg coefficient mRNA binding site on small subunit tRNA bidning sites and peptide canal on large subunit tRNA sites 0 A site aninoacyltRNA O P site peptidyltRNA O E site Exit Ribosome is a ribozyme rRNAs fold into highly compact structure 0 Catalytic site is 23S rRNA O Ribosomal proteins bind the outside of rRNAs First stage small and large ribosomal subunits assemble around an mRNA that has an activated initiator tRNA positioned at the start codon in the ribosomal P site I G protein involved in activating initiation factors I Peptidyl chain elongation in eukaryotes GTP used for conformational change as peptidyl change moved and as ribosome moves along RNA I Continues until reaches stop codon I Ribosome is a ribozyme O O O O O rRNAs fold into highly compact structure Ribosomal proteins bind the outside of rRNAs Catalytic site in 23S rRNA part of large subunit of ribosome Large molecule During first stage of translation small and large ribosomal subunits assemble around an mRNA that has an activated initiator tRNA positioned at the start codon in the ribosomal P site In eukaryotes this process is mediated by a special set of proteins known as eukaryotic translation initiation factors tRNA methionine starts at P site new tRNAs progress through APE sites until stop codon a termination factor binds to stop codon GTP hydrolysis of termination factor causes ribosome to disassemble Some antibiotic inhibit protein or RNA synthesis I Tetracycline blocks binding of aminoacyl tRNA to Asire of ribosome I Streptomycin prevents the transition from initiation complex to chain elongating ribosome I Chloramphenicol blocks the peptidlyl transferase reaction on ribosomes I CycloheXimide blocks the translocation reaction on ribosomes I Fidaxomiacin Blocks initiation of RNA chains by binding to RNA polymerase MesselsonStahl experiment showed that DNA followed the semiconservative mechanism was how DNA was replicated Incorporation of new nucleotides to growing chain Two step reaction base pairing and covalent bond Energy from incoming nucleotide 5 I 3 direction DNA template dNTP s dATP dCTP dTTPdGTP Enzymes helicase toposimerase single stand DNA binding protein clamp DNA dependent DNApolymerase primase RNA polymerase 0 Primer RNA polymerases do not need it I Order of following enzymatic activities 00000 0 RNA polymerase 0 DNA polymerase I RNase 0 DNA polymerase 0 DNA ligase 0 DNA polymerase many sub types I Highly processive up to 1000 bpsec I Two catalytic sites 0 Polymerizing or P site 0 Error rate of 1 error every 1X10quot5 bases 0 Double check mechanism base pairing leads to conformational change conformational change leads to bound formation 0 Editing or E site 0 3 I 5 exonulecase O Stalling mechanism in P brings misincorporated nucleotide to E site 0 Decreases error rate 100 times to 1 error every 1X10quot7 bases 0 5 I 3 0 If chain growth proceeded in opposite direction proofreading mechanisms would terminate chain elongation 0 Results in asymmetrical replication forks I Leading vs lagging strand I Origin of replication AT rich sites because only 2 H bonds instead of 3 with GC located throughout chromosomes 0 Both directions I Clamp needed at origin of replication what DNA pol binds to O Allows DNA polymerase to allow procession to continue without disassociation or slipping 0 PCNA preinitiation process for replication 0 Single stranded binding protein keeps replication bubble open 0 DNA polymerase high error rate can introduce mutation s into the genome 0 Error rates with both P and E site 1 for 1X10quot7 bases Human genome 32X10A9 I Approx 320 error per cell cycle Error made during DNA replication must be corrected to avoid mutation errors fixed during replication do not become fixed in the genome errors fixed after replication may introduce mutations 50 of the time 0 Recognition of template and daughter strand one is right one is wrong 0 Strand directed mismatch proofreads newly synthesized DNA strand Further decreases error incorporation 100 times to 1x10quot9 bases error rate in human genome 34 bases per round of replication Most common types of DNA damage Depurination guanine hydrolyzed off sugar no base at that position Deamination cytosine converted to Uracil Pyrimidine dimers consecutive thymine form dimers rather than pairing with adenosine DNA repairs Base excision repair repairing GT mismatches caused by the chemical conversion of cytosine to uracil or deamination of 5methyl cytosine to thymine must recognize which strand is wrong and which is right 50 chance of replacing wrong side 0 DNA glycolyase cut out incorrect base 0 APEl endonucleose cut out sugar 0 DNA pol put in correct base ligase seals nicks Mismatch excision repair eliminates base pair mismatches and small insertions or deletions of nucleotide generated accidently O MSH2 and 6 bind to mismatch location 0 Can tell between daughter and template remove section of nucleotides Gap repair by DNA polymerase and ligase I Predisposition to a colon cancer results form loss of function mutation of one copy of either the MLH1 or the MSH2 gene which are essential for DNA mismatch repair Nucleotide excision repair for thymine dimers Mutations in any of at least seven different genes XPA through XPG lead to inactivation of this repair system causing xeroderma pigmentosum a hereditary disease associated with a predisposition to cancer 0 Individuals with this disease frequently develop melanomas and squamous cell carcinomas if their skin is exposed to the UV rays in sunlight 0 Double strand breaks can be repaired rapidly but imperfect 0 When both stands break no intact strand can guide proper repair 0 Quick fix better than nothing no homologous end joining results in loss of nucleotides I Better alternative mechanisms to fix double strand breaks and other errors homologous recombination allows awless repair of double strand breaks other mismatch errors I Holiday Junctions homologous strands cut exchange and ligation branch migration resolution either heteroduplexes that are recombinants or non recombinants I DNA repair and generation of genetic diversity Meiosis 3202014 0 Molecular Genetic Techniques 0 Ampicillin resistance gene codes for betalactamaid breaks down enzyme that inhibits bacterial growth 0 Restriction enzymes enzymes found in different bacteria first one EcoRl I Identify palindromic sequence and makes a cut I Sticky ends I Recombinant DNA molecules can be formed in a test tube use restriction enzymes to cut and input genes into plasmid I Bacteria produce restriction enzymes methylate s own restriction sites to protect against self 0 Preparing to make a genomic DNA library from yeast need DNA ligase and restriction endonuclease 0 cDNA library would need reverse transcriptase I DNA cloning in a plasmid vector insert vector containing cloned DNA fragment into E coli grow E coli many multiples of gene 0 cDNA libraries can be screened with a radiolabeled probe to identify clone of interest only coding sequence goes from mRNA to DNA 0 Gel electrophoresis I Separates DNA molecules of different lengths I Once run on a gel can isolate and do things 0 PCR amplify DNA regions 0 Denature DNA with heat anneal primers elongate primers repeat 0 Sequencing methods use uorescently labeled dideoxynucleosides I Sanger method dNTPs incorporated into growing chain ddNTPs uorescently labeled when incorporated into DNA sequence stops DNA polymerization because no way to form phosphodiester bond 0 Series of fragments at the end of each fragment a uorescent ddNTP separate by length sequence Southern blot can detect specific DNA fragment in a complex mixture of restriction fragments Run fragments on gel transfer from gel to nitrocellulose membrane hybridize with labeled DNA or RNA probe Autoradiogram Northern blot used to study gene expression by detection of RNA or isolated mRNA I Observe cellular control over structure and function by determining the particular gene expression levels during differentiation morphogenesis as well as abnormal or diseased conditions 0 Hybridization of probe RNA to RNA on a nitrocellulose membrane 0 From a northern blot of an mRNA of interest probed With a fulllength cDNA probe 0 Relative amount of mRNA and size of mRNA In situ hybridization can detect activity of specific genes in Whole and sectioned embryos I Attach probetag to antibody I See Where genes are expressed and When I Good for developmental processes DNA microarray analysis reveal differences in gene expression under different experiment conditions I Spot turns green if gene decreases in cells after serum added I Spot turns red expression of that gene increases in cells after serum addition Yellow present in both cells I How expression patterns change under particular conditions Common inherited diseases for research I Sickle cell anemia cystic fibrosis phenylketonuria TaySachs Huntington s Hypercholesterolemia Duchenne muscular dystrophy Hemophilia Transgenic mice insert gene into egg it recombines into mouse DNA insert eggs into surrogate transgenic mouse baby Knock out genes in specific cell types I loxPCre recombination system gene function disrupted When Cre expressed cuts out gene I Cre viral endonuclease that cuts DNA out put loxP on either side of gene to remove Cre Will cut out section in between loxP sections I RNA interference small inhibitory RNAs design to be complimentary to particular mRNA sequence bind to and block translation of mRNA 0 Morpholino modify gene expression hybridized to mRNA CRISPRs clustered regularly interspaced short palindromic repeats I Viral nuclease found in bacteria can be targeted to a particular sequence 0 Enzyme Cas9 0 Small guide RNA that Will bind to a particular regularly interspaced short palindromic repeat DNA repair mechanisms will fill that in by homologous recombination 0 Used to knock out but also used to repair genes so that they cut out nonfunctional genes and input functional through homologous recombination I Can target cell type and sequence 0 Mice With disease that cause cataracts target gene involved in specific cells cut out non functioning and stick in functioning one time fix can be done in embryonic stem cells inheritable 0 Yeast genomic library can be constructed in a plasmid shuttle vector that can replicate in yeast and E coli 3252014 Structure of genes and chromosomes Histone proteins spool around which DNA wraps to bind it I 30 nm fiber I loops of 30 nm fiber higher order chromatin inaccessible to transcriptional machinery Protein coding regions of DNA approx 2 introns 25 regulatory regions 13 heterochromatin 8 not yet sequenced repeats 52 Alu sites transposable elements retro transposons inserted into different locations in genes sometimes disruptive and can result in inherited disorders Breast cancer Ewing s sarcoma hemophilia diabetes mellitus type II Familial hypercholesterolemia Besides duplicated protein coding genes and tandemly repeated genes eukaryotic cells contain multiple copies of other DNA sequences in the genome referred to as repetitious DNA 0 Of the two main types of repetitious DNA the less prevalent is simple sequence DNA or satellite DNA which constitutes about 6 of the human genome and is composed of perfect of nearly perfect repeats of relatively short sequences I Caused by backward slippage I Inheritable criminal identification paternity determination Transposable elements jumping genes DNA sequences that move within genome creates or reverses mutations alters genome size 0 Large fraction of the Cvalue of eukaryotic cells I Cvalue amount of coding DNA compared to total amount of DNA Generally considered noncoding TEs are important in genome function and evolution 0 DNA transposons cut out and inserted elsewhere cut and paste no net increase in DNA retrotransposon copied using RNA polymerase reverse transcription inserted elsewhere in genome copy and paste net increase in DNA 0 Insertion of transposons can generate spontaneous mutations which can in uence evolution 0 Unequal crossing over between homologous mobile elements at different chromosomal locations leads to exon duplications gene duplications and chromosomal rearrangements that can generate new combinations of exons 0 Subsequent divergence of duplicated genes leads to members of gene families with distinct functions 0 Inclusion of anking DNA during transposition also results in the movement of genomic DNA to another region of the genome 0 This can result in new combos of exons and new combos of transcriptional control regions Most eukaryotes genes involved in metabolism plant has most genes involved in metabolism 0 Number of genes does not account for difference in complexity what accounts number of cell types cell interactions etc 0 Genome size good indicator of number of genes but gene density is higher in lower eukaryotes Chromosomes 0 Single enormously long linear DNA molecule 0 Human genome 32X10A9 nucleotides in 24 chromosomes 0 DNA packed by proteins into chromatin 0 Association between proteins and DNA to form densely packed chromatin I ionic strength of medium can condense more or less 00 0 DNA organization 0 Compaction vs accessibility DNA 2 m cell nucleus 6 micrometers in diameter I Zig zag association of nucleosomes DNA and histomes with another histone to tightly pack I Nucleosomes 8 histones 2 copies of each DNA 2 turns 147 bp linker DNA 1090 bp 0 Can be dissociated with high salt I Histones can be modified have tails that stick out of nucleosome rich in lysine serine arginine histidine and threonine 0 Can be bound and modified by I Phosphorylation I Methylation I Acetylation I Ubiquitination I How accessible DNA is O Nontranscribed genes are less susceptible to DNase I digestion than active genes because retained in condensed state 0 Heterochromatin vs euchromatin actively transcribed and silent DNA regions can be distinguished within nucleus I Hetrochromain includes centromeres and telomeres as well as inactive genes stains darker I Euchromain less compacted state Most transcribed regions of DNA stains lighter I Histones methylated by H3 trimethylated at lysine 9 HPl will bind to methylated tails forms zig zag conformation of heterochromatin I Histone acetylation dissociation of HPl from histone 3 tails 0 Levels of DNA compaction I Chromatin remodeling complexes loosens or tightens DNA to make genes accessible or inaccessible to transcription energetically expensive I Histone modifications I Histone code pattern of acetylation and methylation for gene accessibility 0 Histone modification enzymes regulate chromosome condensation O Histone acetyl transferase HATS O Histone deacetylation complex HDAC 0 Additional proteins bind to acetylated histones and maintain chromatin in a closed state 0 The biological outcome associated with histone methylation depends on the site that is modified Each site of methylation has different surrounding amino acid context which allows the binding of distinct codereader proteins It is the binding of different downstream effector proteins that gives rise to different biological outcomes 0 Boundary elements prevent spread of activesilent chromatin states barrier to prevent formation of heterochromatin prevents heterochromatin from spreading sequence of bases I Changes in chromatin structures are inherited after replication 0 Organization of chromosomes in nucleus pattern of organization chromosome territories characteristic regions of the nucleus different between cell types but the same for all cells in a cell type 0 Juxtaposition can bring genes involved in a certain function closer together variation in gene expression patterns 0 Standard DNA replication leads to loss of DNA at 5 at the end of each strand of linear DNA molecule because of need of RNA primer 0 Telomeres condensed heterochromatin at end of chromosome I Get shorter with each round of replication buffer to loss of protein coding genes I Telomerase uses RNA template to elongate telomeres active during development but then shuts off 3272014 0 Transcriptional Control of Gene Expression 0 Most cells contain the same DNA except mature B and T cells not expressed in all tissue 0 Cloning Dolly and Copycat 0 Reprograming of differentiated cells into induced pluripotent stem cells I Indirect programming turn cell to iPSC and from there turn it into other cells I Direct programming skip iPSC step 0 Cells differentially express a subset of their genes 0 Housekeeping genes necessary for cell growth common across all cells I DNA replication I DNA transcription I Protein synthesis I Metabolism enzymes 0 Cell specific genes I Oxygen carriers I Hormone synthesis I Neurotransmission etc I Overview eukaryotic O Chromatin in condensed state where gene not accessible to polymerase 0 Proteins involved in acetylating and methylation to allow structure to unwind H1 histone falls off followed by methylation and acetylation 0 At particular sites transcription factors bind to promoter sequence on gene enhancer sequences usually up stream sometimes downstream I Overview prokaryotic lac operon 0 Genes turned on and off in response to environmental cues O Lactose not present no need for beta galactosidase O Lactose present and low levels of glucose glucose is preferential to metabolize CAPwith cAMP inhibition of repressor protein lac genes transcribed 0 Eukaryotic gene expression can be controlled at several different steps 0 Transcriptional control I Transcription is controlled by Binding of proteins to specific DNA sequences 0 Proteins transcriptional regulators 0 DNA sequences 0 Promoter O Operators prokaryotes O Enhancers eukaryotes binding of factors to enhancers activates transcription upstream and downstream 0 Regulatory gene sequence 0 Transcriptional regulator 0 DNA binding domain 0 Activatorrepressor domain 0 Homeodomain 3 alpha helixturn helix I Bind to sequences of DNA recruit other proteins to initiate transcription 0 Zinc finger domain 1 alpha 1 beta zinc atom O Leucine zipper 2 alpha helices from different proteins I Dimers more stable 0 Transcription factor interactions diversify gene regulation 0 Same class of proteins homodimers vs heterodimers 0 Different class of proteins cooperation I Two transcription factors bind together to a certain sequence 0 Different base pairs in DNA can be recognized from the helix grooves 0 Enhancer elements are generally distance and orientation independent where as basal promoter elements such as the TATAA box are both distance and orientation dependent 0 Transcription switches tryptophan repressor O Repressible system tryptophan binds to repressor turns genes off 0 Transcription switches Catabolite activator protein 0 Induce or activate gene expression 0 Binding of metabolite cyclic AMP cAMP activate CAP 0 High cAMP low glucose turn on genes involving metabolism of other sugars 0 Transcriptional Switches lac operon O Lactose present binds to repressor and removes from sequence can be transcribed 0 Eukaryotic transcription factors can recruit RNA polymerase from a distance 0 Activators recruit polymerase directly or indirectly open chromatin DNA bending proteins scaffolding proteins transcription binding complex 0 Repressor sabotage pol Recruitment directly or indirectly close chromatin 0 DNA looping model proved because biotin ends connects enhancer to Beta globin via avidin Beta globin still expressed RNA can t scan over avidin must loop over 0 Eukaryotic gene activators can direct local alterations in chromatin structure 0 Chromatinremodeling complex histone acetylation remodel nucleosomes to allow transcription factors mediator and RNA polymerase to get to DNA 0 Combination of proteins regulate eukaryotic gene expression 0 Complex 0 Single transcription regulators can activate numerous eukaryotic genes 0 DNA replication 0 Mitosis 0 Muscle differentiation O O O MyoD transcription factor that regulates transcription of many other transcription factors causes formation of muscle cell Almost any cell can be turned into iPSC via Oct4 Sox2 and Nanog Regulatory proteins activated causes formation of different cell types 0 Maintenance of gene expression profiles through cell division 0 0 Positive feedback loops transient signal turns on expression of protein A remains in cytoplasm remembered Maintenance of chromatin structure condensation of one of the X Barr body inheritance of the pattern of X chromosome condensation direct inheritance of chromosome condensation DNA methylation methylation of cytosine remembered by cell enzymes methyltransferase remember methylation pattern of CG sequence and will restore those 0 RNA Processing Control after transcript is made 412014 Riboswitch self regulating mRNA that undergo conformation changes 0 Conformational change in response to metabolite such as guanine that causes transcription to hault 0 Particularly common in bacteria Untranslated region of mRNA s can control their translation in numerous ways 0 Translation repression proteins forms hair pin repressor protein that prevents ribosome 0 Hairpin repression structures allow proteins to be made 0 Heat shock proteins elevated temperature GC bonds break proteins made 0 Antisense RNA 0 After transcription posttranscriptional processing 0 G cap poly A tail cleave exons 0 Binds to proteins move in and out of nucleus 0 Which modification to mRNA occurs in the cytoplasm O Deadenylation every time message translated some of the polyA tail degraded 0 Degradation 0 Polyadenylation for small messages keeps it stable 0 Eukaryote specific RNA splicing removes internal introns from exons Mature mRNA will be exported to cytoplasm Alternative splicing different ways of hooking up exons Introns intervening sequences Exons expressed sequences Exons can be alternative spliced different proteins made OOOOO Electron microscopy of mRNAtemplate DNA hybrids shows that introns are spliced out during premRNA processing Location of splice sites exonintron junctions in a premRNA can be determined by comparing the sequence f genomic DNA with that of the cDNA prepared from the corresponding mRNA How recognized what to take out what to put together 0 Frequency of occurrence of certain base at certain site At 5 end of intron 100 frequency GU also at branch point A and at 3 endAG Pyrimidine rich region 39 Mechanism of splicing 0 Moderately conserved sequences mark intron boundaries 0 2 transesterification reactions 0 No input of energy required 0 Carried out by spliceosome 0 Exon Junction complex 0 Lariat structure spliced out intron 0 Splieceosome 5 small nuclear RNAs U1 U2 U4 U5 U6 uracil rich 0 610 proteins small nuclear ribonucleoproteins snRNP O recognize consensus site at end of intron and exon complimentary basepairing recruitment of proteins 0 roughly size of ribosome around 170 proteins including 100 splicing factors in addition to proteins associated With 5 snRNPs 0 Exon recognition through cooperative binding of SR proteins and splicing factors to pre mRNA 0 Average length of an exon in the human genome is around 150 bases Whereas average length of intron is around 3500 0 Info for defining splice sites that demarcate exons is encoded Within the sequences of the exons 0 Self splicing introns 0 RNA first served as both catalytic and replicative molecule 0 Some bacteria and some protists no energy involved just transesterification Approx 13 of all hereditary diseases are thought to have a splicing component 0 Mutations of a splice site resulting in loss of function of that site premature stop codon loss of exon or inclusion of an intron 0 Mutation of splice site reducing specificity result in variation in thee splice location causing insertion or deletion of amino acids disruption of reading frame 0 Displacement of a splice site leading to inclusion or exclusion of more RNA than expected resulting in longer or shorter exons Role of alternative splicing in perception of sound 0 Hair in ear calcium channel depolarization stimulates auditory nerve basal lamina tuned to different frequencies location of signal Where depolarized interpreted as pitch 0 Different isoforms produced in different parts of ear take different vibrations to be opened up discriminate pitch gene is the same but spliced into different isoforms RNA editing of apoB premRNA 0 Apolipin protein B Carboxyl terminal binds to low density lipo proteins 0 In liver cells ApoBlOO O In intestine same gene but after RNA transcribed edited to earlier stop codon ApoB48 I RNA editing after transcription change in base sequence I ApoB 48 important in moving fats but doesn t have sticky carboxyl end Model of transporter passage through NPC 0 Get RNA from nucleus to cytoplasm anchor proteins anchor to membrane scaffold proteins filaments in cytoplasm and nucleus many proteins involved 0 Nuclear transport proteins bind to mRNA and transport mRNA through nuclear pore 0 FG nucleoproteins stick out into center of nuclear pore phenoalinine and glycine non polar transport proteins take message through non polar areas 0 Can be localized in cell to different places For a proteincoding gene posttranscriptional control of gene expression can be regulated 0 By controlling stability of mRNA in cytoplasm 0 Control rate of translation 0 Control where its localized too microRNAs control gene expression 0 2225 nucleotides long produced from a longer RNA precursor endogenous to the cell 0 Assembled into an RNA induced silencing complex RISC O Complementary to sequences in 5 or 3 UTR of target mRNAs O RISC binding to an mRNA complementary to micoRNA reduces translation or destroys the target mRNA RNA interference RNAi destroys foreign doublestranded RNA viruses 0 Defense mechanism against viral dsRNA O dsRNA cleaved by dicer and incorporated into RISC complexes 0 in plants RNAi actively transfers from cell to cell base pairing with target RNAs distinguishes miRNA and siRNA 0 miRNA not complete hybridization siRNA complete I miRNA translation inhibition incomplete hybridization I siRNA RNA cleavage complete hybridizatioin miRNA processing 0 dicer cuts microRNA loop of hairpin 0 mouse limb with knock out of dicer no dicer no miRNA silencing mutant formation silencing important in development concentration of an mRNA is a function of both its rate of synthesis and its rate of degradation decapping pathway deadenylation dependent pathway endonuleolytic pathway degradation Relatively short lifespan in cytoplasm Cdk mRNA cytokines mRNA in immune cells Which of the following events are catalyzed by an RNA 0 Translation and splicing ribosomes and spliceosomes also contain proteins but RNA is catalytic function 432014 Fluid mosaic model of bio membranes Well established Intrinsic proteins membrane proteins peripheral membrane some anchored to cytoskeleton Selective barrier Transduce information Mechanically elastic Compartments Variable composition Allows maintaining constant electrochemical gradient electrolyte density etc Internal organization of cells and attachment to cytoskeleton very important 0 Different membranes have different protein and lipid composition re ects function 0 00000000 Bilayer structure 0 Hydrophobic inside hydrophilic outside 0 Micelle hydrophobic interior 0 Liposome bilayer aqueous center Faces of cellular membranes 0 Cytosolic interior cytoplasm 0 Exoplasmic outside 0 With ER Golgi endomembrane system exoplasmic within 0 Faces of cellular membranes are conserved during membrane budding and fusion 0 Membrane lipids O Glycerol two carbons linked to fatty acids one carbon linked to phosphate and variable portion of head group phosphatidyl choline ethanolamine sereinelinositol Plasmalogen Sphingolipids Steroids cholesterol membrane buffer retains uidity and rigidity depending on temperature Fatty acids I Saturated proton on each carbon single bonds C I Unsaturated CC 0 Can be cis kink or trans no kink 0 Fluorescence recovery after photo bleaching FRAP experiments can quantify the lateral movement of proteins and lipids within the plasma membrane 0 Phospholipids move very quickly laterally 0 Label phosphate groups with uor bleach certain area on cell membrane with membrane look for uorescent intensity after starts to recover up to 50 shows recovery because unbleached move into I Some of the phospholipids are attached to proteins and do not move lipid rafts why it s only 50 0 Diffusion coefficient dependent on size phospholipids move faster than proteins 0000 0 Fluidity of membrane depends on its composition 0 The closer and more regular packaging of tails the more viscous less uid the membrane is I Number of hydrocarbons I Number of double bonds I Cholesterol stiffens membrane stabilizes I Phospholipids pack together through Vander Waals stronger for saturated unkinked tails 0 Plasma membrane loses and gains membrane area via endo and exo cytosis composition of those membranes involved differ how do they keep up composition 0 Lipids move laterally but can t move across bilayer without certain enzymes specific for type of phospholipid that they do ippases specific scramblases ip any 0 Asymmetrical distribution of lipids in plasma membrane 0 Pinositol stays on cytoplasmic side activates pathways in cells 0 Pserine and Pethanolamine on inside 0 Pcholine glycolipids and spingomyelin on outside each part of membrane has own composition 0 Fluid and assymetrical I Fluid is measure of diffusion in 2D cannot readily ip remain in the mono layer unless actively transferred by an enzyme I Protein interactions and anchoring to microfilaments allow uid structure to remain a structure 0 Synthesis of Fatty acids 0 Growing fatty acid chain linked to coenzyme A O Sequential addition of two acetyl units at a time 0 Additional enzymes form double bonds 0 Glycerol phosphate fatty acyl coA tails embed in membrane choline or other group attached to head ippase to ip if needed on cytosol side to begin 0 Enzymes either cytoplasmic or embedded in membrane Cholesterol 0 Important component of membranes 0 Precursor to other molecules bile acids vitamin D steroid hormones cholesterol esters modified proteins Functions of membrane proteins 0 Transporters move molecules across membranes 0 Anchors anchor to cytoskeleton and exterior ECM without ability to anchor to basal lamina can metastasize O Receptors 0 Enzymes Proteins associate with membranes in different ways 0 Span membrane hydrophobic area that is thermodynamically favorable to interact with lipids 0 Single pass transmembrane protein 0 Mulipass membrane proteins alpha helices forming around channelpore inside hydrophilic outside hydrophobic for transmembrane part Anchor glycoportiens anchored in membrane 0 Lipid end of fatty acid can be anchored by protein directly attached or cholesterol with protein attached Charged residues can orchestrate assembly of multimeric membrane proteins 0 Associatedissociate and move for example tyrosine kinase receptor Plasma membranes of cells contain combinations of glycosphingolipids and protein receptors organized in glycolipoprotein microdeomains termed lipid rafts O Specialized membrane microdomains compartmentalize cellular processes by serving as organizing centers for the assembly of signaling molecules 0 In uence membrane uidity and membrane protein trafficking and regulate neurotransmission and receptor trafficking 0 More ordered and tightly packed than the surrounding bilayer but oat freely in membrane bilayer Membrane spanning need to be helices or beta sheets because need to have polar groups tucked in to prevent from interacting with phospholipids Plasma membrane is reinforced by the cell cortex 0 Red blood cells shapes because of cytoskeleton and hemoglobin Visualizing movements of proteins at cell membranes label human cell with uor and a mouse system with different uor measure how long takes for equal diffusion diffusion coefficient of different things Cell surface is coated with carbohydrates mostly outside of cell 0 Proteins imbedded in membrane 0 Glycoprotein receptor signaling Difference between blood types type A has extra sugar different gal than B Recognition of cell surface carbohydrates on neutrophils is the first stage of their migration 0 Neutrophil bind to certain lectins cause shape of neutrophil to change can move through to site of infection Molecules large and small have to traverse lipid membranes to maintain cell viability O Gases diffuse membrane permeable 0 Small unchaged polar molecules membrane permeable or slightly permeable 0 Large uncharged polar molecules ions charged polar molecules impermeable 39 Some way to get across Membrane transport proteins 0 ATP powered pumps against concentration gradient active transport 0 Ion channels move with concentration gradient do not require energy facilitated transport require receptor to allow though I Ligand gated voltage gated O Uniporter symporter 2 separate molecules one down gradient the other up antiporter exchange molecules Multiple membrane proteins function together in the plasma membrane of metazoan cells 0 NaK pump 3 Na out for 2 K in pump against gradient ATP needed electrogeneic moves ions across gradient establish potential because net movement of positive charges out 0 Potassium moves down concentration gradient through potassium channel coupled to NaK O Nalysine symporter lysine move against gradient Na moving with gradient Facilitated diffusion movement down a concentration gradient protein helps across cell membrane no energy used Cellular uptake of glucose by GLUT proteins exhibits simple enzyme kinetics O Glucose transporter in blood cells GLUTl much lower Km than that in liver cells GLUT2 O Glucose taken in even at very low levels because virtually no glucose in cytoplasm metabolized right away 0 Less efficient transporter in liver because liver take up glucose and store as glycogen to store later Gradient of movement of calcium from outside to inside 0 Operational model of Ca2 ATPase in the SR membrane of skeletal muscle cells 0 ATP to pump Ca into ER SR mitochondira to sequester concentration in cytoplasm low I When Ca released casues things 0 In muscle actin myosin contractions 0 Activate kinases Proton pump against concentration gradient electric potential 0 Lysosomes decreas pH Cystic fibrosis transmembrane regulator allows chloride to move from one side to the other sodium might also move out 0 Osmotic gradient draws water across 482014 Moving Proteins into membranes and organelles To maintain order different intracellular processes are segregated inside the cell 0 Segregation sites where different processes take place DNARNA synthesis energy production detoxification cell sorting 0 Main function of membraneenclosed compartments how are proteins segregated to the appropriate compartment I Organelles with different sets of proteins and enzymes compartmentalize I Proteins are imported into organelles by three mechanisms 0 Nuclear import signals sequence 0 Folder protein 0 Transport proteins 0 Transport across membrane signal sequence 0 Unfolded protein 0 Protein translocator 0 Transport by vesicles signal sequence 0 Folded protein 0 Transport proteins I Cytoplasmic proteins those involved in glycolysis and others that remain in cytoplasm do not need signal sequence in default state I Secreted proteins as synthesis starts signal sequence on Nterminus of protein that comes out of ribosome Translation stops until signal sequence binds to signal recognition particle take to receptor on ER open pore in ER protein extruded from ribosome into lumen I Targeting system to mitochondria or chloroplast must cross both membranes can also be inserted into outer or inner membrane or into intermembrane space Signal sequence direct proteins to the correct organelle usually on Nterminus except for peroxisomes and some nucleus proteins Signal sequence not removed from peroxisome or nucleus proteins Structure of Rough ER studded With ribosomes typical of secretory cells ribosome attached to ER translocanes pore complexes open through series of protein interactions allowing for protein to be injected into ER lumen O No energy involved beyond What is needed for protein synthesis Translation and translocation occur simultaneously completed proteins cannot be put into microsomes Protein export through ER 0 Synthesis of protein targeted for export membrane secretion I Protein synthesis in the ER protein modifications in ER protein modifications in Golgi O Vesicular transport I ER to golgi export and maintenance of ER proteins I Golgi apparatus and protein sorting I Exocytosis and endocytosis Single or multi pass trans membrane proteins remain anchored through hydrophobic anchor synthesis continues but the remainder remains on cytoplasmic side Cotranslational translocation synthesis starts in cytoplasm Beginning signal sequence Will bind to Signal recognition protein Which Will at as ligand for SRP receptor associated With translocon channel for protein to be put through signal sequence cleaved by signal peptidase but protein continue to go through translocase fold in lumen ER membrane proteins classified via single pass or multiple pass Whether inner part is amino or carboxyl group Whether internal part is very short compared to outside or not GPIlinked proteins Single pass proteins stop transfer anchor sequence that causes dislocate from channel and embed self in ER membrane Positive charges on amino terminal side causes amino side to be on cytoplasm side signal anchor sequence near bottom of translocon sequenced continues in such a way as COO end up on ER lumen side Tail anchored protein Cterminal tail associated With complex that takes to channel in ER membrane causes carboxyl end to be imbedded in membrane happens after complete translation GPI anchored proteins protein synthesized With hydrophobic tail most of protein in ER lumen part in lumen removed and attached to GPI anchor Once embedded in membrane modified Unfolded protein response UPR trigger the production of chaperones misfolded proteins bind to receptor in ER lumen activate transcription regulator to move into nucleus and activate transcription of chaperone genes 0 Misfolded proteins must be dislocated to cytoplasm if can t be fixed because proteolytic machinery for ER proteins is in cytoplasm Protein import into mitochondrial matrix general import pore pore in inner and outer membranes 0 Heat Shock proteins proper folding I Transport into mitochondria is post translational heat shock prevents from folding so doesn t fold wrong in cytoplasm I To get into intermembrane space Nuclear pore complex at different levels of resolution 0 FG motifs hydrophobic residues that stick out act as gatecloud to prevent movement of hydrophilic molecules I Nuclear import nucleus localization signal binds to importin in cytoplasm that has hydrophobic amino acids on outsides can move through channel and carry protein with it I Once inside nucleoplasm release cargo and importin moves back into cytoplasm to bring another protein in dissociates and moves out with GTP I Already transcribed and folded before into nucleus also happens in Golgi because folded in ER lumen and sent into Golgi 4 1514 Vesicles pinch from membranes Each vesicle carry different proteins Each vesicle has a different destination Vesicle fuses with target membrane Vesicular movement through endomembrane system pinch off modified in ER and golgi fuse to cis side of golgi closer to ER I move through medial golgi to trans golgi 0 Form endosomes lysosomes O Exosomes release content secretion In pulse chase experiment give a short pulse of radioactive leucine to pancreatic beta cells the insulin producing cells in vertebrates After a short time you chase the radioactive leucine with an excess of non radioactive leucine fix the cells after 3 7 37 117 minutes after the nonradioactive luecine was added to the media 0 Find the radioactive leucine using autoradiography I Initially near ER starts to coalesce in golgi begins to put together zymogen granules insulin holding secretory vesciles moves out of cell Protein transport through secretory pathway can be visualized by uorescence microscopy of cells producing a GFP tagged membrane protein 0 Plasma membrane protein uorescence decay over time in different areas to follow protein movement Luminal side of vesicle stays on the inside through golgi and becomes outside plasma membrane part Cytosol side remains cytosolic Vesicle budding is driven by the assembly of a protein coat 0 Coats proteins I Provide a scaffold that establishes membrane curvature I Interact with cargo proteins or cargo protein receptors to provide enrichment of certain protein in the bud I recruits others I Clathrincoated vesicles 0 Golgi to endosome 0 Plasma membrane to endosome 0 Outside of vesicles direct secretory vesicles to plasma membrane and to endosomes in endocytosis I COP coated vesicles 0 ER to Golgi 0 Within Golgi cistema I Golgi to ER 0 Anterograde and retrograde traffic Coated vesicles are formed by polymerization of cytosolic coat proteins onto a donor membrane to form vesicle buds Eventually pinch off from the membrane to release a complete vesicle After vesicle release the coat is shed exposing proteins required for fusion with the target membrane 0 Budding initiated by recruitment of GTP binding protein Vesicle buds can be visualized during in vitro budding reactions spontaneous reaction between coat membrane and buds other proteins needed to pinch off Types of coated vesciles O COPII vesicles transport protiens from ER to Golgi O COPI vesicles retrograde transport proteins between golgi cisternae and from the cisgolgi back to the ER 0 Clathrin vesicles transport proteins from plasma membrane and the trans golgi network to late endosomes Small GTP binding proteins belonging to GTPase superfamily control polymerization of coat proteins initial step in vesicle budding 0 After vesicles are released from the donor membrane hydrolysis of GTP bound to ARF of Sarl triggers disassembly of the vesicle coats O Coat proteins not only important in budding off and movement but in stabilizing Golgi structure without them proteins normally found in Golgi never get there Short amino acid sequences target protein to specific organelles 0 ER resident proteins carry KDELsequence 0 KDEL receptors move between ER and Golgi O Receptors only bind KDEL proteins at high H If you were to remove the ER retrieval signal from protein disulfide isomerase soluble resident of the ER lumen where would you expect the modified PDI to be located outside of the cell its gradual ow out of the ER to the Golgi apparatus would not be countered by its capture and return to ER Follow default pathway out of the cell Vesicle docking depends on tethering and SNARE proteins 0 Snare complex for docking fuse and release cargo 0 Recognition specific proteins for specific vesicles and suck tethering protein binds to Rab Rab proteins GTPbindin proteins regulate docking of the veislces with correct target membrane Each RAb binds to a specific Rab effector associated with target membranetethering protein VSNARE in a vesicular membrane binds to cognate tSNARE protiens in the target membrane inducing fusion of the two membreanes The SNARE complex is disassembled in an ATP dependent reaction Functions of Golgi apparatus 0 Stacks of ever maturing membrane enclosed sacs moving down as matured becoming cis then medial then trans then vesicle 0 Protein maturation I Proteolytic cleavage I Sugar modification I Protein modifications 0 Vesicle sorting I Retrograde I lysosome I Late endosome I Constitutive secretion I Regulated secretion upon a signal 0 How is it that soluble proteins in the ER can be selectively recruited into vesicles destined for the Golgi I Soluble ER protein that are diestined to reside in other membrane organelles or to be secreted are bound bttransmembrena cargo receptors The cytosolic domains of these cargo repcotors bind to the COPII coats on the veislces that form on the ER membrane incorporating the cargo recpotrs into COPII coated vesicles 0 Anterograde transport through golgi occurs by cistemal maturation often addition of sugars 0 Two models of protein trafficking through Golgi I Cistemal maturation I Vesicular transport 0 Vesicle budding clathrin coated vesicles transport selected cargo molecules as clathrin sarts binding vescle starts forming dynamin pinch off vesicle once released clathrin uncoats and naked transport vescle is formed 0 Endocytic vesicles and veislces that bud from transGoli have netowek of coat of AP adaptedr protein complexes and clathrin heavy and light chain 0 Pinching off clathrincoated vesicles require dynamin forms collar around neck of veislce budn and hydrolyzes GTP 0 M6P receptors in trans Golgi network bind proteins with M6P residues 0 Direct their transfer to late endosomes where receptors and their ligand protiens dissociate O Receptors are recycled to Golgi or plasma membrane and lysosomal enzymes are delivered to lysosomes 0 Endocytic pathways budding off of the plasma membrean to form an intracellular veislce O Phagocytosis cellular eating 0 Pinocytosis cellular drinking regulation of plasma membrane side I Receptro mediated endocyrosis receptors on plasma membrane protiensmolecules bind accessory membranes bind to cytosolic side of receptor pinches off clathrin disassembled 0 LDL will then fuse with endosome and lysosome free cholesterol 0 Endocytosed macromolecules are sorted in endosomes I Lysosomes are the principal site of intracellular digestion O Sacs of hydrolytic enzymes 0 Function in acidic contions O Membrane contains ATPdependent H pump and transporters I Acid hydrolases work best at low pH won t kill cell if breaks I Lysosomes are the final destination for material degradation autophagy destruction of internal organelles that are no longer functional 4 17 14 0 Cell signaling 0 Intracellular signaling components relay amplify integrate and distribute signal 0 Around 50 of drugs targeted to affect receptors 0 Extracellular signaling 0 Endocrine signaling hormone secretion into blood by endocrine gland Paracrine signaling adjacent Autocrine signaling target same cell Signaling by plasma membrane attached proteins When signaling pathway modifies a protein already present activity is changed immediately when partway modifies gene expression there will be a delay corresponding to the time ti takes 0 O O O for the mRNA and protein to be made for the cellular levels of the protein to be altered sufficiently to invoke a response which would usually take and hour or more 0 Cell surface receptors 0 Ion channel coupled receptors 0 Gprotein coupled receptors 0 Enzyme coupled receptors 0 Simple signal transduction pathway involving one kinase and one target protein binding of signal molecule causes activation of kinase which phosphorylates and activates other molecules I transcription 0 Stopped by nuclear phosphatases inactivate kinases removing phosphate monomers to dissociate stops reaction I GTPase switch proteins cycle between active and inactive forms 0 GEF guanine nucleotide exchange factor activator protein calls for GTP O GAP GTPase accelerating protein inactivator protein 0 Second messengers on inside of cell 0 cAMP proteinkinase A O cGMP activates protein kinase G I opens cation channels in rod cells 0 DAG activates protein kinase C O 1P3 opens Ca2 channels in ER 0 Amplification of an extracellular signal accomplished by cascade amplification at each level from small initial concentration I G protein coupled receptors activate heterotrimeric Gproteins with intrinsic GTPase activity 0 Receptor bind ligand 0 Anchored in membrane cytoplasmi domain associated with a G protein 0 G protein alpha beta and gamma gamma and beta binds to signal molecule I GTP binds to alpha I alpha breaks off and activates effector falls off come back together If affinity for GDP lowered without change to affinity of GTP would be constantly active 0 Dissociated heterotrmeric Gproteins have different intracellular targets in cardiac beta goes and binds potassium chanel as well as alpha binding and activating effector which could be adynyl cyclase creating cAMP which can be degraded to AMP when finsished 0 Alpha subunity can activate adenyly cyclase and phospholipase C I CAMP pathway enzyme phosphorylation transcription factor phosphorylation CREB I Phospholipase C IP3PKC pathway PKC protein phoslphorylation Calmodulin other Ca2 depedent enzymes I cAMP can stimulate metabolism and gene transcription adrenaline system causes glycogen breakdown and causes transcription of target gene rapid energy source short tem and long term I cAMP differeing affects in different cells different proteins in cascade O 0 1P3 pathway triggers rise in intracellular Ca2 I Synthesis of second messengers DAG and 1P3 from P1 PI clipped DAG remains in membrane 1P3 becomes soluble in cytosol I Ca2 signaling indirectly triggers many biological processes I Ca2NOcGMP pathway and relaxation of arterial smooth muscle 0 Acetylchline binds to GPCR phospholipase C I 1P3 I Ca2calmodulin I NO synthase leads to relaxation of muscle cells I Viagra 0 Several common cellsurface receptors and signal transduction pathways 0 Receptor associated kinase 0 Cytosolic kinase 0 Protein subunit dissociation 0 Protein cleavage 0 Receptor tyrosine kinase hormone O J AK cytokine receptor 0 Exists as monomers in membrane cytoplasmic side has kinase activity 0 4222014 I Kinase bind ligand I conformational change dimerization activation of kinase activity in cytosolic domain auto phosphorylate attracts scaffolding proteins that bind to are activated elicit response I Most RTK s activate Ras following ligand binding to RTK or cytokine receptors 0 Ras anchored to cytoplasmic side of membrane upon activation other proteins recruit to RTK scaffolding activates Ras which binds GTP and is activated signaling downstream I many pathways 0 MAP kinase pathway several kinases activated in cascade by Ras 0 MAP mitogen activated protein mitogen is cytokine that stimulates cell growth and cell divison O Eventually changes protein activity and gene expression 0 MAP can lead to transcription of fos gens regulation of cell cycle and cell growth I RTK s activate the PI3 KinaseAkt signaling pathway that promotes cell survival activate PI 3 kinase phosphorylated inositol phospholipid protein kinase El protein kinase I Activates Akt which frees Bcl2 from Bad allowing it to inhibit apoptosis I Cytokine RTK s prolactin directly activate transcription factors activate STAT 5 forms dimer joins other transcription factors for milk protein genes I milk production I Protein kinase networks integrate information to control complex cell behaviors GPCR and RTK interact Some intracellular singaling proteins serve to integrate incoming signals Secretion of insulin in response to rise in blood glucose ucose transported into cell via GLUT2 metabolized to make ATP and pyruvate ATP activates TP sensitive K channel move out of cell down gradient depolarization open Ca channels into cells cause insulin congaing secretory vesicle to fuse to membrane and release insulin Galpha protein render GPCR pathway constitutively active if Galpha cannot hydrolyze GTP Which of the following steps in an intracellur signaling pathway amplifies the signal synthesis of secondary messenger activation of protein kinase receptor activation of G protein A loss of function mutation in the type of TGFbeta receptor is found in about 25 of colon cancers Drug that has which of the following effects counteract mutation I Constitutive activation of Smad 3 0 Eukaryotic Cell cycle 0 0 00000 Cells reproduce by duplicating their content and dividing in two G1 grow S DNA replication G2 grow more M divide Checkpoints ensure proper sequence of events only moves forward I G1 checkpoint is environment favorable I G2 checkpoint is all DNA replicated Is all DNA damage repaired I Checkpoint in mitosis are all chromosomes properly attached to the mitotic spindle Prophase breakdown of nuclear envelope formation of spindle fibers Metaphase lining up 0 chromosomes Anaphase separation of chromosomes Telophase and cytokinesis splitting of cells Cycle dependent kinases regulate cell cycle I G1 CDKS prepare cells for S phase I Ubiquination of G1 cyclins into S pahse I SPhase CDKS activate DNA replication 0 Mitotic CDKs induce mitosis CyclinCdk complexes function in different phases 0 G1 checkpint arrest the cell cycle if environmental conditions make cell division impossible or if the cell passes into G0 for an extended period of time G1SCdk complex commit the cell to a new cycle CDK4 and Cyclin D SCdk complexes promote S phase and block it in G2 CDK2 and cyclin E and A O M Cdk complexes trigger entry into itosis CDK1 and Cyclin B I Appears in mitosis phosphorylates lamin and leads to nuclear envelope breakdown during early mitois I cycBCdk1 will be destroyed during mitosis to allow formation of new nuclear envelop during telophase 0 cell cycle control systems depend on cyclically activated protein kinases Cdks cyclin concentration cycles rises to critical point in mitosis before declining rapidly to interphase Cdk concentration constant 0 Levels of Cdk activity change during the cell cycle in part because cyclin levels change during the cycle Different ccyclineCdk complexes trigger different steps in the cell cycle 0 Cell cycle according to oscilationg cyclinecdk and apc activity a cyclin promotes synthesis of the next cyclin that in turn promotes destruction of the previous one I APC anaphase promoting complex get cell out of mitosis 0 How are Cdks regulated I By cyclin synthesis and destruction I By phosphorylation OO O I Cdk activity is regulated by phosphorylation and dephosphorylation 0 Two phosphorylation sites activating and inhibitory 0 Double phosphorylation or no phosphorylation inactive 0 Phosphorylation only at activating site dephophorylation of inhibitory cite activated I By binding to CDK inhibitory proteins CKIs 0 Progression through the cell cycle requires a cyclin to bind to a Cdk because binding of cyclin to Cdk is required for Cdk enzymatic activity 0 Other modulators of cyclineCdk activity I some kinases inhibit activity I Wee1 inhibits phosphorylation I Cdc25 phosphatases reverses the effect 0 Balance between two I Cdk inhibitory proteins CKIs inhibit direct binding I Cyclical proteolysis ensure termination of cyclincdk activity ubiquitination of cyclin marks it for destruction 0 APC destroys mitotic cyclins contains E3 ubiquitin ligase I Regulated Prteolysis 0 APC targets M cyclin ends of mitosis I SCF ubiquitin ligase targets CKI Mitogen receptor RTK pathway sends signal through pathway to start transcription of genes nvolved in cell division 0 Genes usually silent until signal inactivates Retinoblastoma protein allow transcription factor to bind to genes RB protein called master brake of cell cycle describe how the Rb protein acts as a cell cycle brake How is the brake released in mid to late G1 to allow cell to proceed to S phase 0 Master break because binds transcription factor necessary for transcription of genes for the S phase When phosphorylated transcription factor released and can transcribe genes Proteins that inhibit Cdks can arrest the cell cycle at specific checkpints 0 G1 damaged DNA unfavorable environment will go into G0 liver cells in G0 for long time contact inhibition S damaged of incompletely replicated DNA G2 damaged or incompletely replicated DNA M chromosome improperly attached to mitotic spindle 0 All involve inhibition of Cdks Sphase cyclinCdk complexes initiate DNA replication once per cycle 0 Unphosphorylated origin recognition complex ORC binds to origins O Cdc6 binds to ORC in G1 0 Mini chromosome maintenance proteins Mcm assemble to complete pre replication complex 0 SCdk phosphorylates Cdc6 and subsequently ORC and Mcm I Degradation of phosphorylated Cdc6 prevents multiple replication 0 ORC remains phosphorylated until end of cell cycle 0 After replication Mcm is exported from nucleus How does SCdk help guatantee that replication occurs only once 0 Phosphorylates the Cdc6 protein marking it for destruction Activation of Mphase CylinCdk complexes MCdks triggers entry into mitosis O Mcyclin levels rise in G2 0 MCdk is held in check by phosphorylation until Cdc25 phosphatase produces small amount of active MCdk O MCdk activates Cdc25 inactivates Weel and thus promotes a burst of active MCdk I Positive feedback on Cdc25 by active MCdk I Active MCdk also inactivates Weel very rapid increase in MCdk activtity OOO Cohesins hold sister chromatids together forms ring around chromatids 0 During anapahse have to separate cohesins must be cleaved I Cleavage controlled by enzyme separase held inactive by securin which is activated by Cdc20 and APC ubiquinates securin I Cyclin and securing must be destroyed in order for anaphase to take place Cytoskeleton carries out both mitosis and cytokinesis O Microtubules of mitotic spindle O Contractile ring for cytokinesis DNA damage checkpoints prevent replication of damaged DNA 0 Activation of p53 usually at low concentratins rapidly degraded phosphorylation stabilizes I Regulatory protein binds to region of DNA that activates transcription of p21 gene which is a Cdk inhibitory protein inactivate Cdk complex until damage fixed Activity of G1 S MCDKs is out of phase 0 G1 cyclin accumulates as a result of the activation of the AktmTOR cell growth and survival pathway I G1 cyclinCdk functions induces Scyclin expression stops M cyclin degradation I Activated Tor prevents G1 cyclin degradation 0 Scyclin begins to acculate activated by G1cyclinCdk complex I Pre replication complex assembles at the origin of replication complex but is not active I Triggers DNA replication and degradation of Glcyclin 0 Prevents from going backwards once committed to replication 0 G1 CDK blocks ubiquitinase that degrades MCyclin which begins to accumulate as well I Completion of DNA replication activates McyclinCDK complex I MCyclinCdk complex activates different steps in mitosis and its own destruction What cellular mechanisms ensure the passage trough the celly cycle is unidirectional and irreversible What molecular machinery underlies these mechanisms 0 cyclin for one step activates synthesis of cyclin for second step which degrades cyclin from previous step 4242014 I Apoptosis programmed cell death is a normal cellular function used in 0 Development to remove excess cells 0 Adulthood to maintain the proper cell number 0 Disease to remove damaged cells 0 Map out process of apoptosis in C elegans I Apoptosis and Necrosis O Necrosis spilling their contents and triggering in ammatory response 0 Apoptosis results in orderly destruction of cell and packaging into membrane bound vesicles sculpting and development I Apoptosis is a quick and neat process blebbling breakup of nuclear envelope cell fragmentation engulfed and destroyed by phagocytic cell I Apoptosis is mediated by an intracellular proteolytic cascade procaspase activation by cleavage link up caspace 9 which activates other caspase in amplifying cascade cleave nuclear lamin and cytosolic protein 0 Extracellular activation of apoptosis activation of apoptosis from outside the cell extrinsic pathway killer lymphocyte with Fas ligand caspase cascade apoptosis 0 Intracellular activation of apoptosis injured mitochondria proteins open channels ions ow in and let cytochrome c leak out cytochrome c binds to Apafl which binds to caspaases forms apoptosome activates caspases caspase cascade O Regulated by Bc12 family of intracellular proteins I Good antiapoptotic prevents cell death 0 Bc12 blocks Bax Bak activity I Bad proapoptotic promotes cell death 0 Bax Bak release cytochrome C I Ugly pro apoptotic blocks good guy activity 0 Bad inhibit Bc12 I Loss of function mutation in which of the following might be found in cancer cells Bax 0 Survival factors suppress apoptosis by regulating Bc12 family members Akt regulation I Survival signal activates PI 3 kinase 1P3 and phosphorylated DAG activate Akt inactivates Bad cannot sequester Bc12 0 Survival factors suppress apoptosis by upregualting Bc12 family members transcription regulation 0 Changes in cell membrane composition labels the cell as ding cells membrane becomes permeable no longer a barrier inconsistent with life0 I Change in membrane composition phosphatidyl serien is no longer restricted to cytoplasmic side I Exposed phosphatidyl serine is a tag labeling a cell or phagocytosis 0 Animals cells require extracellular signals to survive proliferate and grow 0 O O O 0 Survival factors promote survival by preventing apoptosis Mitogens stimulate cell division by overcoming intracellular breaking mechanisms that block cell cycle progression Growth factors stimulate increase in cell size Signals are transduced through cell surface receptors Mitogens stimulate cell division by releasing the G1 to S transition breaks I Myc is a regulator gene that codes for a transcription factor thought to regulate 15 of all genes mutated Myc is found in many cancers constitutively expressed unregulated expression of many genes I Overactivity of cell cycle stimulators normally triggers arrest and apoptosis Normal somatic cells have limited growth potential I Progressive increases in Cdk inhibitors I Progressive shortening of telomeres in cells that do not express telomerase Growth factors stimulate synthesis and decrease degradation of macromolecules I A signal transduction through posphatdiyl inositol pathway I Kinase cascade leads to increase translation I Some factors stimulate both growth and cell cycle progression Neural pathfinding depends on cell growth and apoptosis Nerve growth factors can in uence both rate and direction of growth I Cell growth initially responds to gradients of growth factors I Not all nerve cells reach their target I Neruons that do not make it or make it late die Myostatin mutants decrease apoptosis in muscle tissue Normal cells need both mitogens and anchorage to enter a new cell cycle I Stop growth by contact inhibition form mounds by pumping new medium across cells ow of medium causes growth I Signals from the substratum increase probability of entering S phase extracellular anchoring is important 0 Cancer arises from violations of the basic rules of social cell behavior proliferate without control 0 00000 Resisting cell death Sustaining proliferative signaling Evading growth suppressors Activating invasion and metastasis Enabling replicative immortality Inducing angiogenesis I Defects in DNA replication I Defects in DNA repair I Defects in cellcycle checkpoint I Mistakes in mitosis I Abnormal chromosome numbers I Directing growth of new blood cells to cancer cells to provide nutrients and oxygen Protooncogenes genes normally part of cell survival growth and so on gain of function mutation never turned off unregulation Tumor suppressor genes inhibit cell survival or proliferation loss of function mutations unregulation and proliferation Caretaker genes repair and prevent DNA damage loss of function mutations allow mutations to accumulate Which of the following are encoded by proto oncogenes I Bel2 resistanct to apoptosis I Cyclin D self sufficient for growth and insensitive to anti growth signals I Telomerase limitless replicative potential Tumors evolve by repeated rounds of mutation and proliferation Key behaviors of cancer cells I Reduced dependence on survival mitogenic and growth factors I Less prone to apoptosis I Reactivate telomerase I Increased mutation rate I Lack specific celladhesion molecules invade tissue I Survive outside their normal environment I Incidence of human cancers increases as a function of age multiple mutations I Tumor appearance in female mice carrying either one or two oncogenic transgenes shows the cooperative nature of multiple mutations in cancer induction I Energy production in cancer cells by aerobic glycolysis with or without oxygen most glucose metabolized into lactate I Effects of oncogenic mutations in protooncogenes that encode cellsurface receptors 0 Ligand independent phosphorylation activation signaling even without ligand I Loss of p53 abolishes the DNA damage checkpoint 0 P53 direct cells to apoptotic pathway arrest of cell cycle activation of repair enzymes 0 Without p53 cancer cells proliferate I Loss of telomeres normally limits number of rounds of cell dvison 0 Indefinite replication embryonic or stem cell 0 Limited replicatin somatic cell 0 Breakagefusionbridge cycle chromosome instability apoptotic cell death senescent cell 0 Persistant growth but also chromosome instability cancer cells I Therapeutic targeting of the hallmarks of cancer 0 Up regulate CKIs 0 EGFR inhbitors I Wharberg effect inhbitors 0 Proapoptoic molecules 0 PARP inhibitors involved in repair of DNA 0 Inhibitors of VEGF signaling inhibit angiogenesis VEGF vascular growth 0 Inhibiting HGFcMet anti metastasis 0 Selective antiin ammatory drugs 0 Telomerase inhibitors 0 Immune activating anti CTLA4 mAb avoide immune detruction
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