Cell bio.doc

19 Pages

Biological Sciences
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Tanya Da Sylva

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Poly A tail plays a role in the degradation UTR 1) MRNA made exported to cytoplasm Reaches 30 pts—poly A nuclease is actively recruited, chops the tail up decapping enzyme is recruited Exonucluease from 5’ to 3’ degrades message Exosome collection of proteins Factors that affect stability Active factors that shut off RNA 1) produced by own cells called miRNAS  bind to a site in the 3’ UTR (untranslated regions through complementary base pairs) Induce degradation of target mRNA 1)Interfere with translation—active translation 2) inhibit after initiation- post initiation inhibiation 3) cause mRNA to degrade—inhibition of inti 4) mRNA degradation 2) RNA interference RNAi—petunais discovery C elegans—(Mello and Fire) injected worms with sense RNA, anti sense and double stranded RNA Double stranded RNA worked Double stranded rna taken up by cells and degraded the RNA it was complement to but was SPECIFIC  genetic immune system 1) Cells produce transcript that forms double bonded regions of complenmatrity 2) BOTH strands are cleaved by DICER—cleaved into very short fragments with OVERHANGING ENDS 3) Associated with pre-RISC that has an argonaute protein— removes PASSENGER strand—NOT complementary to mRNA we want to degrade—its recognized by pre-RISC 4) Guide strand—associates with RISK, brings to target RNA, target RNA is cleaved 5) RNAi- target RNA is cleaved- and degraded 6) Mirna—translation can be blocked, mRNA can be destabilized, block ribosome a. Differences due to complementarty is NOT exact, needs additiona structures in MiRNA DNA replication Three ways Semiconservative conservative Dispersive Figured out semi using radionucleotides 1) parental dna was incubated with heavy and transferred to light media 1 generation all strands were HYBRIDS—heavy dna and light (parental and new) nd 2 generation—more and more light- more strands being made, hybrids don’t go away— Eukaroytes BDRu Original had thymine introduce BDRU Now all cells are radioactive Both chromotides have brdu and one strand with thymidine One cells dividing-each daughter cells will get ONE strand of parental DNA Temperature sensitive --replicate until switched to higher temperature and then they stop Higher—NONE permissive or restrictive temperature 3) create protein in ecoli- cause they can fit in small space, can create in vitro systems still heavily studied in prokaroyotes Diagram replication --origin of replication, time for cells to replicate genome proteins bind to origin and start process, DNA happens bidirectionally parental forks at when replication is JUST starting Supercoiling 1) negative supercoiled—slightly underwound 2) positive supercoild—overwound Toposiomerase—change topology of DNA Change supercoiled state by creating break that allow DNA to twist around themselves DNA gyrase—cleaves BOTH strands of DNA, attached to one strand and release tension Requires ATP 1) Circular—w/template 2) straight—w/e template 3) break in the middle of the strand 3’ Lagging strand --against movement of replication fork little pieces are ligated together Replication Fork --parental helix is undergoing strand separation Geometry --Angles of bonding are similar geometry drives a conformational change Mispairing doesn’t have the same geometry Can occasionally occur Mistakes --wrong nucleotide incorporate—STALLS the polymerase incoroporated base pair, its recognized something has gone wrong newly synthesized strand is sepearted forms new 3’ terminus that can be cleaved polymerase tries again Eukaryotes Genomes are replicated Eukaryotes --multiple origins of replication AT ARS—multi protein complex form (ORC) Replication of yeast --Licensing factors bind to origin and help intiate replication requires activation factors --protein kinases—main steps of activating replication DNA REPAIR --detect subtle distortions --some damage can be directly repaired, but mostly DNA repair requires excision of damaged DNA 1) FIND damage DNA Global pathway—XPC protein complex --survey entire genome—and happen to be on a damaged basepair Transcription couple—stalled RNA polymerase associated CSB proteins ready to recognize damage base TFIIH --initiation factors for transcription Base excision repair 1) enzymes constantly surveying DNA—global 2) bind to backbone, and rotate nucleotide out of HELIX 2) if nucleotide is right kind of damaged based, cause conformational changed that will cleave base off 3) AP endonuclease—cleaves DNA Cell Division 1) Mitosis- 2) Meiosis M phase—lasts an hour interphase—longer Interphase 1) G1-cell grows, metabolizes 2)S DNA replicated, and chromosomes duplicated 2) G2: after replication, cells grows and prepares to divide M phase --mitosis --cytokinesis splitting of the cell Cell cycle: G1, S, G2- interphase G1- END of mitosis to beginning of DNA replication S- dna replication G2- end of replication to beginning of mitosis Three cell types 1) specialized—lost ability to divide 2) cells don’t divide 3) divide frequently- STEM cells i. stem cells divide daughter cells get the same DNA, but one will get different set of proteins than other 1. one will stay a stem cell 2. DNA the same, but protein and RNA complements are different G1 phase and M phase- premature chromosomal compaction G2 and M phase same thing S phase and M phase—premature chromosomal compaction but DNA was in small bits and sensitive to damage Transition from G2-M was under positive control --some agent telling the cell to go into M phase by fusing together, that agent, diffuse the across the whole cell and force mitotic cell division MPF 1) Kinase—phosphyraltes serine and theroine 2) Cyclin- regulatory subunit *** KINASE***- add phosphates group to other proteins Kinase and cyclin --kinase needs to phosphorylates other proteins to trigger M phase but can only do that with cyclin and concentration spikes as mitosis occurs production of cyclin is linked to cell cycle, concentration increases binds to kinase subunit—causes phorphylation of downstream subunits Cdks --activated those liscensing factors --numerous cyclin dependant kinases in the same way cdc2 gene fission yease CdKS Cdc28 in budding yeast -reponsible for shifts from g2 to S and G2 to M Fission yeast 1) passes START—def replicates DNA Mammals—restriction point Phase just between transition to S phase Replication is going to occur 2) passage through start—requires act of Cdc2 by G1 cyclins 3) g2 to M—by Cdc2 but bound to mitotic cyclins Rapidly degraded by cell—cell moves forward in cycle Cyclin Binding --cycling binds to subunit Cdk -merge of cdh and cyclin---phosphorylates proteins CdK Phosphorylation  phosphateses removes phosphates group single phosphorylated cyclin is active Balance in cell, wee1 and cdc25 determines whether the enzyme is active Balance is determined by other kinases SCF-G1 cyclons APC mitotic cyclins Destruction of mitotic cyclins is needed for cells to exit mitosis Subcellular localization --cell blocks nuclear accumulation fails to initiate mitosis CdK mammals --Cdk1 is the only Cdk required --redundancy—multiple proteins that can take the place of others if they are taken away Cyclins diagram --Normally—during G1 to S transition cyclins D’s and Cd4 and Dk6 Transitionn point—E CDk2 S to G2 –cyclin B/a and Cdk1 Take out all CDks except cdk1—cell cycle still occurs Checkpoints --dna damage critical processes haven’t been completed DNA is damaged beyond repair, check points can signal cell death or convert to senescene where it no longer replicates Function by -sensors to detect abnormalities—enzymes that detect DNA repair -other enzymes that transmit signal --effect cell cycle 1) atr phosphorylates CHECKpoint kinase 1 2) check point K1—phos cdc25 3) cdc25 binds adapter protein 3) prevents nucle import cdc25 5) cdc 25 removes phosphates from cdK 7) cdk1 inactive M Phase --mitosis—nuclear division --two nuclei identical genomes are created Cytokinesis- cell splits, cytoplasm and organells are portioned to create two separate daughter cells Mitosis --prophase  chromosome compaction—earlier steps supercoiling and compaction requires condensing and topoisomerase II associate with nuclear cytoskeleton form protein scaffold --Doubles of everything Prior to replication DNA is associated with cohesion --once DNA is replicated holds sister chroms together Diagram --red= cohesin G1 =one chromosome replicated—hold together at centromere w/ cohesion Kintochores—out surface of centromeres assembles DURING prophase kinetochores will be site of chromosomes attaching to microtubules Several motor proteins involved in chrom movement reside Centromere forms by DNA Kinetechores around centromere are responsible for movement Centrosome cycle— 1) G1 paired loosely 2) S phase—cetrioles are replicating 2) Procetrioles elongate 3) Beginning of mitosis long enough, separate to opposite poles and now have two centrosomes at opposite ends of cells 4) Microtubule netowwork drive chrom move along Centrosomes separate—microtubles increase in number forming bipolar mitotic spindle Certain types of animal cells lack centrosomes—happens without centrosomes—caused by associated motor proteins Nuclear enveloped and organelles -assemble occurs=cytoplasm --compaction-nucleoplasm Nuclear enveloped must break down—end of prophase Some organelles are intact mitochondria Prometphase --disolution of nuc envelope Mitotic spindle is completed Chromosomes are moved to the middle of cell Sister chromatids, become connected to mircotubles from opposite poles Diagram --altering polymerization rate of either pole equalize length of microtuble Metaphase --metaphase plate --microtubles and mitotic spindle are higly organized 1) Astral microtubules—centrosome to outside body spindle chromo micro—move to poles polar- support structure Diagram 1) astral spindle—faces toward cell membrane 2) chrosomosal spindle fibers attach to kintechore regions, responsible for movement of kintechores 4) polar move toward chromosomes Anaphse Sister chromatids split and move apart --APC-anaphase promoting complex activated at meta and ana transition APC binds to cdc20 or cdh1 APC binds cdc20digest securing marks it marks for destruction, inhibits anaphase inhibitor is gone and anaphase occurs APC cdh1- mitotic cylins for destruction Cyclin can renter into interphase Concentration APC cdh1 or CDC20 flucates with cell life span Destruction of securing releases separases that cleaves subunits that hold sister chromatids together Mitotic cyclines to cdh1—destroys activity of mitotic cdk1 and that’s the end of M phase Choromsomes split in synchrony Microtubules are sho
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