Term
|
Definition
• G1: checkpoint 1
• G2: checkpoint 2
• S: synthesis phase
• M: mitosis phase (cell division)
*cell cycle will stop if any issues w/ 1st or 2nd checkpoints |
|
|
Term
|
Definition
• synthesis phase
• DNA replication (each DNA molecule = chromatid)
• sister chromatid adhesion
• in bacteria, replication & segregation occur in same step |
|
|
Term
|
Definition
• cell division where parental cell is used to form two identical daughter cells
• chromosome segregation
• sister chromatids segregated to opposite poles of cell; each daughter cell thus rcvs identical info when cell divides
• Phases: PMATC (prophase, metaphase, anaphase, telophase)
• cytokinesis actually splits one cell into two |
|
|
Term
|
Definition
• first checkpoint
• makes sure DNA not damaged before S phase
• parental cell preparing for DNA replication |
|
|
Term
|
Definition
• 2nd checkpoint
• makes sure DNA fully replicated before M phase
• cell preparing for chromosome segregation |
|
|
Term
| 2 major events during cell cycle |
|
Definition
• DNA replication
• chromosome segregation |
|
|
Term
| What happens during checkpoints - G1 & G2 |
|
Definition
• allow cells to check to ensure necessary materials and energy present to proceed to next phase
• makes sure DNA not damaged |
|
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Term
|
Definition
| pair of DNA duplicated molecules |
|
|
Term
|
Definition
| individually copied DNA molecule |
|
|
Term
| sister chromatid cohesion |
|
Definition
| cohesin holds together sister chromatids until segregation |
|
|
Term
| microtubule-organizing centers |
|
Definition
• centrosomes (animal cell) or spindle pole bodies (yeast/fung)
• form poles at opposite ends of cells
• microtubules pull sister chromatids towards poles, preparing for cell division |
|
|
Term
| sister chromatid separation |
|
Definition
| cohesion ring destroyed, sister chromatids segregate to opposite poles of cells |
|
|
Term
|
Definition
• G1, S & G2 phases
• when mitotic division not occuring
• DNA is not compact or condensed |
|
|
Term
|
Definition
• long DNA strands wrap into highly condensed chromosomes
• nuclear envelope breaks down
• microtubule organizing centers migrate to opposite poles of cell; signals metaphase |
|
|
Term
|
Definition
• microtubules (long fibers) attach to each kinetochore of chromosome pair (centromeres when chromatids) to organizing centers on either side
• two chromosomes form bivalent attachment
• microtubules pull sister chromatids towards each pole
• chromosomes align in middle of cell because cohesin rings still surrounding them |
|
|
Term
| bivalent attachment (metaphase) |
|
Definition
| • two kinetochores of sister chromatid pair are attached to microtubules of opposite sides (poles) of cell |
|
|
Term
| monovalent attachment (metaphase) |
|
Definition
• only one kinetochore of sister chromatid pair is attached to a microtubule on one side of cell (pole)
• results in uneven segregation of chromosomes into daughter cells |
|
|
Term
|
Definition
| proteolytic destruction ® signals anaphase |
|
|
Term
|
Definition
• cohesion between sister chromatids lost (via proteolysis)
• chromosomes migrate to opposite poles of cell |
|
|
Term
|
Definition
| • new nuclear membranes form around each sets chromosomes before cells split, final step mitosis |
|
|
Term
|
Definition
| cell divides into two daughter cells |
|
|
Term
| cell cannot perform cytokinesis |
|
Definition
| one cell will have two nuclei with duplicate chromosomes |
|
|
Term
| cell cannot break apart cohesin rings |
|
Definition
| anaphase affected and cell could not segregate chromosomes to opposite poles from center |
|
|
Term
|
Definition
| proteins that DNA winds around before tightly compacting |
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|
Term
|
Definition
• assembly to form compact DNA to fit into body
• histone forms a spool with DNA wrapped around it
• 8 histones (two each of H2A, H3, H2B & H4) form 1 core
• assembled from histone protein subassemblies
• H3 & H4 form tetramer subassembly
• 2 H2A & H2B dimers form two subassemblies
• each histone protein has N-terminal tail
• DNA wraps around each histone core 1.65 times (147bp in length) |
|
|
Term
|
Definition
• 3billion bp = 1 meter long if stretched out
• to fit into body, DNA wound into 23 pairs of chromosomes |
|
|
Term
|
Definition
• DNA must be duplicated
• two new daughter strands rapidly reassembled into nucleosomes
• old histones distributed between two new DNA strands
• new histones brought in to fill in gaps |
|
|
Term
| N-terminal tail on histone |
|
Definition
• provide guide for DNA to wrap around histone core
• emerge between DNA strands & create groove (like screw)
• force DNA to twist around histone in left-handed manner |
|
|
Term
| what binds DNA to core of histone |
|
Definition
|
|
Term
| H3 & H4 tetramer binds what part of DNA? |
|
Definition
| middle & both ends of core DNA |
|
|
Term
| H2A and H2B dimers bind to what part of DNA? |
|
Definition
| binds DNA between middle and ends |
|
|
Term
| how are multiple nucleosomes connected? |
|
Definition
• adjacent nucleosomes connected by short stretches of DNA called linker DNA
• fifth histone, H1 protein, is linker histone that binds linker DNA |
|
|
Term
|
Definition
• stimulates first level of chromatin packing, formation of 30nm fiber
• binds both end linker DNA and middle of nucleosomal DNA, bringing adjacent nucleosomes into close proximity
• larger than other histones at ~21 kD |
|
|
Term
|
Definition
• less condensed form of chromatin
• "beads on a string" |
|
|
Term
|
Definition
• more condensed version of chromatin
• one of two structures (might differ between species) ®
• solenoid
• zig-zag |
|
|
Term
|
Definition
| • nucleosome disks stacked on top of one another; forming helix; linker DNA packed inside |
|
|
Term
|
Definition
| • requires longer linker DNA because of how the nucleosomes cross over one another from opposite sides (may be preferred form for species w/ longer linker DNA) |
|
|
Term
| highly condensed mitotic chromosome |
|
Definition
• 30nm fiber must fold even further
• fold into large loops, held together by nuclear scaffolding proteins at the base of each loop
• then folded again to form condensed chromosome |
|
|
Term
|
Definition
• associated proteins must be reassembled on each daughter DNA molecule
• nucleosomes must be partially dissassembled to allow replication machinery to pass
• histone synthesis & modification occurs |
|
|
Term
| histone synthesis & modification |
|
Definition
• H3:H4 tetramers remain bound to one of two daughter DNA duplexes at random
• H2A:H2B intact but released into pool of free dimers, surrounding replication fork
• H3:H4 tetramers from old nucleosomes form new nucleosomes on daughter DNA after replication fork passes
• because amt of DNA doubled, more histones synthesized and recruited
• histone chaparones guide to DNA behind replication fork |
|
|
Term
|
Definition
• Asf1 & CAF-1 work together to bring new H3:H4 tetramer to assembly site that didn't get old tetramer, where PCNA ring is
• NAP-1 brings two H2A:H2B dimers to each new strand (each gets one old dimer and one new dimer)
• histone modifications carefully preserved
• essential to survival of each daughter cell |
|
|
Term
|
Definition
• proliferating cell nuclear antigen rings
• sliding clamp proteins that tether DNAP to DNA during replication; left on new DNA to serve as markers |
|
|
Term
| modified old & new histones |
|
Definition
• modified old histones can recruit enzymes that modify nearby new histones in same way
• i.e. enzymes w/ bromodomains bind to acetylated histones & acetylate nearby new histones |
|
|
Term
| Why is packing DNA into chromosomes important? |
|
Definition
• it allows DNA to fit into cell
• protects DNA from damage
• only packaged DNA can be transmitted to daughter cells
• chromosomal DNA very stable (naked DNA not)
• chromosome has overall organization to each DNA molecule |
|
|
Term
| Why is organization of DNA important? |
|
Definition
• organization regulates: gene expression & recombination
• recombination btw parental chromosomes - generates diversity seen w/ different individuals of any organism |
|
|
Term
| molecular mass of eukaryotic chromosome |
|
Definition
• half of mass is made up of proteins
• most are histones and some are nonhistones |
|
|
Term
|
Definition
• given region of DNA w/ its associated proteins
• these proteins help to compact DNA |
|
|
Term
|
Definition
• majority of DNA-associated proteins
• small, basic proteins
• have high contect of (+) charged amino acids
• < 20% of residues in histones are lysine or arginine |
|
|
Term
|
Definition
• include DNA-binding proteins
• they regulate transcription, regulation, repair & recombination of cellular DNA |
|
|
Term
| size properties of chromosomes |
|
Definition
• human cell contains 3x109bp per haploid chromosomes set
• avg thickness of each bp = 3.4Å
• if stretched out = ~1010Å or 1m
• diploid stretched out = 2m |
|
|
Term
|
Definition
• typically only has one complete copy of chromosome packaged into nucleoid
• portions of chromosome present in two and sometimes four copies during rapid division |
|
|
Term
|
Definition
• independent, circular DNA
• not essential for bacterial growth
• carry genes that confer desirable traits to bacteria (antibiotic resistance, etc) |
|
|
Term
|
Definition
• two copies of each chromosome
• most eukaryotic cells are diploid |
|
|
Term
|
Definition
| two copies of given chromosome (one from each parent) |
|
|
Term
|
Definition
• only single copy of each chromosome
• involved in sexual reproduction |
|
|
Term
|
Definition
• more than two copies of each chromosome
• some organisms - majority of adult cells in polyploid state |
|
|
Term
| global genome amplification |
|
Definition
• hundreds or thousands of copies of each chromosome
• allows cell to generate larger amts of RNA & proteins |
|
|
Term
|
Definition
• specialized polyploid cells
• produce thousands platelets that lack chromosomes but essential to human blood (maintains high metabolism) |
|
|
Term
|
Definition
| chromosomes contained w/i membrane-bound organelle |
|
|
Term
| genome size, number of genes |
|
Definition
• correlates with organism's complexity
• generally though, it's the number of genes, not necessarily genome size
• prokaryotic cells have genomes <10Mb
• single-cell eukaryotes <50Mb
• complex protozoans >200Mb |
|
|
Term
|
Definition
| increased complexity = less gene density |
|
|
Term
|
Definition
• decrease gene density
• discontinuous, protein-coding regions
• takes up more than 60% of human genome
• either unique or repeated |
|
|
Term
|
Definition
• interspersed non-protein-coding regions
• removed from RNA after transcription
• 95% of average protein-coding gene (5% actually encodes) |
|
|
Term
|
Definition
| removes introns from RNA before translation = mature mRNA |
|
|
Term
|
Definition
• 25% of intergenic DNA
• regions of DNA required to direct/regulate transcription
• nonfunct relics, mutant genes, fragments, pseudogenes, ori's |
|
|
Term
|
Definition
| coordinate gene expression - direct/regulate transcription |
|
|
Term
| mutant genes & fragments arise from… |
|
Definition
| simple random mutagenesis or errors in DNA recombination |
|
|
Term
|
Definition
• enzymes that copy RNA into dsDNA used by viruses
• where pseudogenes come from |
|
|
Term
|
Definition
• small structural RNAs (maybe >400 in human cells)
• regulate expression of other genes by altering stability of product mRNA or ability to be translated |
|
|
Term
|
Definition
• almost half of genome are repeats; two types:
• microsatellite DNA
• genome-wide repeats |
|
|
Term
|
Definition
• very short, tandemly repeated sequences (<13bp; CACACA…)
• from difficulties in accurately duplicated DNA
• approx 3% of genome |
|
|
Term
|
Definition
• much larger than microsatellite (>100bp, can be 1kb)
• either as single copies throughout genome or clusters
• all forms are transposable elements |
|
|
Term
|
Definition
• sequences that can "move" to diff places in genome
• they multiply and accumulate throughout genome
• rare process in human cells, but now 45% of genome |
|
|
Term
|
Definition
| elements move to new position in genome, often leaving original copy behind |
|
|
Term
| important non-genetic portions of eukaryotic chromosomes |
|
Definition
• origins of replication: direct duplication of chrom DNA
• centromeres: "handles" for movement of chromosomes into daughter cells
• telomeres: protect and replicate ends of linear chrom |
|
|
Term
|
Definition
• sites where DNA replication machinery assembles to initiate replication
• usually 30-40bp apart in eukaryotes
• prokaryotes usually only have one ori |
|
|
Term
|
Definition
• required for correct segregation of chromosomes after replication
• they direct formation of elaborate protein complex, kinetochore
• each chromosome only has ONE centromere |
|
|
Term
|
Definition
| interacts w/ centromere DNA and microtubules (protein filaments) that pull sister chromosomes away from each other into two daughter cells |
|
|
Term
|
Definition
located at the two ends of linear chromosome, bound by several proteins
• proteins distinguish natural ends of chromosome from sites of chromosome breakage & other DNA breaks
• act as specialized origin of replication - allows cell to replicate ends of chromosomes (recruits telomerase)
• portion is in single-strand form and usually TG rich |
|
|
Term
|
Definition
telomeres recruit this DNAP to faciliate end replication
• during replication, lagging strand synthesized as short fragments (okazaki) --> RNAP removed by RNAse H, filled in by DNAPs, then ligated by DNA ligase
• DNAP only able to add to 3' end - even if primase able to synthesize, DNAP cannot replicate DNA when primer removed = short region of unreplicated ssDNA left at end of chromosome
• incomplete sections = end replication problem |
|
|
Term
|
Definition
• incomplete replication of 3' terminus of template DNA caused by exclusive 3' to 5' activity of DNAP
• unreplicated ssDNA leads to loss of genes
• ends of euk chromosomes called telomeres (GT rich repeats) - 3' end extends past 5' end
• most euk use telomerase = protein + RNA (1.5 copies of compliment of telomeric sequence 3'-UAACCCUAA-5')
• telomerase extends 3' end of telomere - does not need template; telomeric RNA serves as template
• can be repeated many times, extending 3' end of chromosome
• DNA replication machinery then extends 5' end of telomere
• DNAP still cannot extend all the way to end of 5'
• telomeric repeats protect by acting as buffer as non-coding DNA & proteins protects degregation of chromosomes |
|
|
Term
| chromosome structure changes |
|
Definition
¨after cell division occurs, chromosome structure altered many times; two states ®
• interphase (chromosome decondensation)
• M phase (chromosome condensation) |
|
|
Term
| SMC (structural maintenance of chromosome) proteins |
|
Definition
• form defined pairs by interacting through lengthy coiled-coil domains ®
• cohesin & condensin
• w/ non-SMC proteins, they form multiprotein complexes that link two DNA helices together
• cohesion links sister chromatids together
• condensin ring w/i individual chromosomes = tigher pack |
|
|
Term
| meiosis (2nd half of euk cell division) |
|
Definition
• specialized to produce cells w/ 1/2 # chrom than parent
• follows DNA replication w/ 2 rounds of chrom segregation
• Metaphase I, Anaphase I
• Metaphase II, Anaphase II ® four gametes (or spores)
• elongated G2 phase
• monovalent attachment occurs in metaphase I
• bivalent occurs in metaphase II |
|
|
Term
|
Definition
• homologous sister chromatids pair ® 4 chromosomes
• chromatids from diff homologs recombine to form link btw homologous chroms ® chiasma
• during metaphase I, two kinetochores from each pair attach to opposite poles (each monovalent) |
|
|
Term
|
Definition
| like mitosis but instead of splitting chromatid pair into 2 cells, 2 sets of 4 chroms split into 4 cells ® dsDNA in each cell |
|
|
Term
|
Definition
| DNA btw each nucleosome ("beads on a string") |
|
|
Term
|
Definition
• DNA most tightly associated w/ nucleosome
• typically only 20-60bp long (diff's from larger structures) |
|
|
Term
|
Definition
| typically associated w/ non-histone proteins for gene expression, replication, recombination |
|
|
Term
|
Definition
• mediates assembly of histone-only intermediates & formation of head-to-tail heterodimers of specific histone pairs
• w/o DNA, core histones form intermediate assemblies in solution
• fold-domain is conserved region in each core histone, w/ 3 a-helical regions separated by 2 short, unstructured loops
• H3 & H4 form heterodimers, then together forms tetramer
• H2A & H2B stay as heterodimers |
|
|
Term
|
Definition
• sites of extensive mods that alter fxn of indiv. nucleosomes
• includes methylation, phosphorylation, acetylation
• protease cleaves the tails, leaving histones intact |
|
|
Term
|
Definition
| approximate twofold axis of symmetry in nucleosomes |
|
|
Term
|
Definition
• DNAP is enzyme that catalyzes synthesis of new DNA
• 3 Domains: Palm, Finger, Thumb |
|
|
Term
|
Definition
• Two things needed for DNA synthesis ®
• dNTPs (deoxynucleoside triphosphates)
• primer:template junction
• each of 4 dNTPs have 3 phosphoryl groups attached to 5' OH of 2' deoxyribose (named a, b, and g phosphates) |
|
|
Term
|
Definition
• primer template junction has two components ®
• template provides ssDNA to be copied
• primer provides free 3' OH at growing end of DNA
• 3'OH of primer attacks a-phosphoryl of incoming dNTP
• leaving group is pyrophosphate w/ b and g
• pyrophosphate (rapid) hydrolysis by pyrophosphatase
provides additional free energy to drive reaction into two
phosphate groups (b and g)
• process can be repeated w/ 3' OH of new dNTP as nucleophile • chemistry requires that DNA be made in polar fashion (extending 3' end of primer strand, so 5' ® 3')
• template strand directs what dNTP is added |
|
|
Term
|
Definition
• Palm: active site for DNA
• correct bp important for catalysis reaction to continue
• Also binds 2 divalent metal ions for DNA polym activity
• once correct bp formed, finger encloses dNTP
• conformational change brings dNTP and primer into correct orientation w/ metal ions |
|
|
Term
|
Definition
• recognizes dNTPs vs rNTPs (even though rNTPs more)
• it can sterically disclude rNTP's because w/ 2'OH, it's too small to fit |
|
|
Term
|
Definition
• metal ion A deprotonate 3'OH of primer, producing oxyanion to attack a-phosphate of incoming dNTP
• metal ion B coordinates (-) charges of b and g phosphates of dNTP & stabilizes pyrophosphate leaving group
• Finger residues also help to ®
• Lys & Arg stabilize pyrophosphate
• Via stacking interactions, Tyr helps hold dNTP in place for
catalysis
• Finger Domain also associates w/ template, turns it 90o to avoid confusion of template vs. primer in palm's active site
• only single template base in active site so palm knows what base to add to primer next |
|
|
Term
| Proofreading (in palm domain) |
|
Definition
• it hydrogen bonds to base pairs in minor groove of new DNA
• h-bonds only form if nucleotides correctly base paired
• if mismatched bp added, replication rate slows
• primer template free to move around exonuclease site
• exo site removes incorrect bp from 3' DNA end site backwards
• primer:template slides back to DNA replication site |
|
|
Term
|
Definition
• no interaction w/ catalysis process
• interacts w/ DNA most recently synthesized
• reduces rate of dissociation btw junction & DNAP
• holds primer:template junction in active site |
|
|
Term
|
Definition
• Parental DNA copied to form two daughter DNA molecules
• leading strand: pulled to right, copied continuously
• lagging strand: copied backwards, discontinous, okazaki fragments; 100-1000bp in length |
|
|
Term
|
Definition
• DNAP has to add to 3' end of lagging, opposite of leading
• therefore, DNAP must move opposite direction, only adding discontinuously in small fragments
• short fragments are okazaki fragments (100-1000bps) |
|
|
Term
|
Definition
• Parental DNA must be copied into two daughter
• enzymes that couple ATP hydrolysis to separation of DNA
• hexameric proteins in shape of ring
• junction btw new separated template strand & unreplicated dsDNA called replication fork
• moves continuously through unreplicated dsDNA |
|
|
Term
| SSBs (single-stranded DNA binding proteins) |
|
Definition
| • bind to ssDNA to stabilize separated strands |
|
|
Term
|
Definition
• as DNA unwinds, twist number decreases
• write number increases = (+) supercoiled DNA
• Topo's remove (+) supercoils = (-) supercoil |
|
|
Term
|
Definition
• specialized RNAP that makes short RNA primers using ssDNA as template
• DNA primase activated by interacting w/ DNA helicase |
|
|
Term
|
Definition
• synthesis of DNA catalyzed by DNAP
• can only add dNTPs to 3' OH of polynucleotide
• bcz DNA antiparallel, one strand synthesized continuously towards repl fork, other is synthesized discontinously away from repl fork
• takes 1 second for DNAP to bind to DNA
• can add up to 1000 nucleotide bases per second |
|
|
Term
|
Definition
| ability of an enzyme to catalyze reactions before releasing substrate |
|
|
Term
|
Definition
• binds to DNAP, holding DNAP & DNA together
• surrounds DNA to increase processivity of DNAP |
|
|
Term
|
Definition
• To complete replication, RNA primers must be removed
• RNAse H degrades RNA bp'd to DNA (H = hybrid for RNA:DNA)
• single ribonucleotide directly linked to DNA removed by exonuclease (changes based on euk vs. prok)
• single-strand gaps left behind by RNAse H are filled in by DNAP's |
|
|
Term
|
Definition
| • nicks btw 3'OH of repair section and 5' phos of replicated section repaired by DNA ligase |
|
|
Term
|
Definition
• two DNAP's to replicate leading/lagging strands often linked in holoenzymes
• trombone model? ssDNA template on lagging pulls through DNAP, allowing DNAP to add nucleotides to 3' end of growing strand
• sliding clamps act as loaders - help DNAP to find primed DNA |
|
|
Term
|
Definition
| multi-protein complex where core enzyme activity (i.e. DNAP) associated w/ additional components that enhance function |
|
|
Term
| Semi-Conservative Replication |
|
Definition
| in each new DNA double helix, one strand is from the original molecule, and one strand is new |
|
|
Term
| Where does prokaryotic replication take place? |
|
Definition
|
|
Term
| Where does eukaryotic replication take place? |
|
Definition
| In the nucleus during S phase of the cell cycle |
|
|
Term
| What is the purpose of DNA replication? |
|
Definition
| Duplicate chromosomes, so that after mitosis each daughter cell will inherit a complete genome |
|
|
Term
| In what type of cells does DNA replication occur? |
|
Definition
| In dividing cells only; non-dividing cells are blocked in G0 and do not progress into S phase, so they do not replicate their DNA |
|
|
Term
| What are the requirements for DNA replication? |
|
Definition
| DNA polymerase, Mg2+, template, primer, and dNTPs |
|
|
Term
| In what direction does DNA replication occur? |
|
Definition
|
|
Term
| What does complementarity mean? |
|
Definition
| For each A on the template strand, there is a corresponding T added to the new strand. The same applies for a G on the template strand. A corresponding C is added to the new strand |
|
|
Term
| What enzyme is reponsible for removing mismatched nucleotides? |
|
Definition
|
|
Term
|
Definition
| Replication proceeds in both directions from central origins of replication (ori) |
|
|
Term
| The type of replication in which the lagging strand is synthesized |
|
Definition
| Discontinuous replication |
|
|
Term
| DNA in a newly synthesized daughter chromosome contains one new strand and one template strand. This is known as? |
|
Definition
| Semiconservative replication |
|
|
Term
| DNA polymerase does what? |
|
Definition
| It is an enzyme that catalyzes the polymerization of dNTPs into DNA |
|
|
Term
| How many types of DNA polymerases do prokaryotes have? |
|
Definition
|
|
Term
| What are the different types of DNA polymerases in eukaryotes? |
|
Definition
| Pol-delta, pol-alpha, pol-beta, pol-gamma |
|
|
Term
| Pol-delta: location, function, processivity, proofreading, use of RNA primer |
|
Definition
• Location: nucleus; function: leading strand synthesis
• processivity: >100,000 bp; proofread: yes; use RNA primer: yes |
|
|
Term
| Pol-alpha: location, function, processivity, proofreading, use of RNA primer |
|
Definition
• Location: nucleus; function: lagging strand synthesis
• processivity: ~180 bp; proofreading: no; use RNA primer: yes |
|
|
Term
| Pol-beta: location, function, processivity, proofreading, use of RNA primer |
|
Definition
• Location: nucleus; function: fill in gaps for repair;
• processivity: ~20 bp; proofreading: no; use RNA primer: no |
|
|
Term
| Pol-gamma: location, function, processivity, proofreading, use of RNA primer |
|
Definition
• Location: mitochondria; function: synthesis of both strands
• processivity: ~8,300 bp; proofread: yes; use RNA primer: no |
|
|
Term
| The cofactor that is required for DNA polymerase activity? |
|
Definition
|
|
Term
| The pre-existing strand read by DNA polymerase is known as? |
|
Definition
|
|
Term
| What are the building blocks of DNA? |
|
Definition
| dNTPs (deoxynucleotides) = dCTP, TTP, dGTP, dATP |
|
|
Term
| What is significant about dNTPs? |
|
Definition
| They lack a 2` hydroxyl group |
|
|
Term
| How is thymine different from uridine? Where is uridine used? |
|
Definition
| It is methylated at the 5` position; uridine is used for RNA |
|
|
Term
| Where does nucleotide polymerization get its energy from? How is this energy stored? |
|
Definition
| Hydrolysis of triphosphate bonds; energy stored in triphos bonds as electrostatic repulsion of negatively charged O2s |
|
|
Term
| T/F: DNA polymerase can bind a 5` phosphate with an incoming 3` -OH? |
|
Definition
| F; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide |
|
|
Term
|
Definition
| It is a strand with a free 3` -OH group; DNA polymerase can only bind a 3` -OH with a 5` phosphate of an incoming nucleotide |
|
|
Term
| What direction does DNA synthesis proceed in? |
|
Definition
|
|
Term
| What direction is the template strand oriented? |
|
Definition
| 3` ® 5`; it is antiparallel to the newly synthesizing strand |
|
|
Term
| Which DNA polymerases have the ability to proofread and determine if the new nucleotide is complementary to the corresponding base of the template? |
|
Definition
|
|
Term
| What happens if an incorrect nucleotide is incorporated? |
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Definition
| The polymerase's 3`-5` exonuclease activity "kicks back", excising the mismatch; pol-alpha and pol-beta lack this activity |
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Term
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Definition
| Continuously synthesized in the 5` - 3` direction |
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Term
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Definition
• The opposite strand synthesized;
• antiparallel to leading strand
• discontinuously synthesized in 5`® 3` direction
• stretches known as Okazaki fragments |
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Term
| How long are Okazaki fragments? |
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Definition
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Term
| What does semiconservative mean? |
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Definition
| It means that each chromatid receives one de nova and one parental strand; DNA replication is always semiconservative, never conservative nor non-conservative |
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Term
| What is the origin of replication (ori)? |
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Definition
| It is the sequence where DNA replication begins |
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Term
| How many ori's do prokaryotic chromosomes have? How many do eukaryotic chromosomes have? |
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Definition
| Prokaryotes = 1 ori per chromosome; eukaryotes = multiple ori per chromosome |
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Term
| What is the general procedure of DNA replication? |
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Definition
| Factors recognize and bind specific ori sequence, unwind DNA, attract components of replication apparatus, apparatus moves down chromosome (away from ori), replicate DNA as it goes; DNA replication is bidirectional, two apparatuses assemble around ori |
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Term
| What is a replicon? What is their average length? |
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Definition
| Region of eukaryotic chromosome that is replicated as unit, from one central ori; length about 200 kb (this is about the length of DNA loops anchored to scaffold proteins, suggesting these proteins may be involved in defining replicon length) |
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Term
| Why is it a benefit to have multiple replicons? |
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Definition
| So that different regions of the genome can be replicated simultaneously; replication begins at the ori in the center of the replicon and extends in both directions until it reaches the end of an adjacent replicon |
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Term
| What is the purpose of helicase? |
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Definition
| Enzyme that unwinds DNA; one of the first factors to bind ori; serves to open double helix so DNA polymerase can replicate strands; is part of replication apparatus |
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Term
| What are single stranded bindng proteins (SSB)? |
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Definition
| Factors that stabilize single stranded DNA by preventing it from winding back into double helix |
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Term
| What does the DNA replication apparatus consist of? |
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Definition
| Helicase, DNA polymerase-delta, pol-alpha, beta-clamp, and primase; two apparatuses assemble, one on each side of the ori, and each moves in the oppostie directions, replicating DNA as they go |
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Term
| What does the beta-clamp do? |
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Definition
| Ring-like protein that wraps around DNA to stabilize association of replication apparatus; required for pol-delta processivity; without clamp = pol-delta only replicates short oligonucleotides (< 200 bp), with clamp = stretches > 100 kb are produced |
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Term
| Strand that is continuously synthesized, in the 5`-3` direction? |
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Definition
|
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Term
| What is DNA polymerase-delta? |
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Definition
| Enzyme that replicates leading strand, reads template one base at a time, incorporates complementary nucleotides and ligates their 5` phosphate to the 3` -OH of the growing strand |
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Term
| What is the overall synthesis of the lagging strand? How is its synthesis overcome? |
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Definition
| Overall synthesis is in the 3`-5` direction (wrong direction); its synthesis is overcome by synthesizing discontinuous short stretches called Okazaki fragments |
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Term
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Definition
| Enzyme that binds unwound lagging strand and transcribes short stretches of RNA (< 15 bp); RNA serves as primer, providing 3` -OH group required by pol-alpha |
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Term
| What is the purpose of DNA polymerase-alpha? |
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Definition
| Enzyme uses RNA primer to synthesize Okazaki fragments of the lagging strand; NO proofreading ability; only synthesizes one fragment at a time while helicase contiues to unwind DNA for next fragment, so lagging strand held in loop |
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Term
| What happens when pol-alpha reaches a primer at the end of a previous fragment? |
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Definition
| Lagging strand is released, primase makes next primer at end of new single stranded region, process is repeated |
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Term
| What are the three enzymes involved with removal of RNA primers from the lagging strand? |
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Definition
| RNAase, DNA polymerase-beta, DNA ligase |
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Term
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Definition
| This enzyme digests any RNA; in replication it serves to remove lagging strand primers |
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Term
| What does DNA polymerase-beta do? |
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Definition
| It fills in the gaps in DNA (~ 20 bp); in replication it fills in the gaps left after RNA primers are removed; N.B. = DNA polymerases can only add nucleotides, they cannot link DNA fragments (pol-beta leaves nicks in DNA) |
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Term
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Definition
| Enzyme that binds any free 3` -OH and 5` phosphates of DNA; in replication it seals the nicks between Okazaki fragments left by pol-beta |
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Term
| What happens as a result of helicase unwinding? What enzyme helps fix the problem? |
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Definition
| Supercoiling increases; topoisomerases helps to restore DNA to its proper level of supercoiling |
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Term
| What happens when the last RNA primer is removed from the end of the chromosome? |
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Definition
| It leaves an overhang that cannot be filled by DNA polymerase; if this didn't get resolved, then every time dividing cells replicated their DNA, the lagging strands would lose a bit of their telomeric sequence, leading to chromosomal destabilization |
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Term
| What enzyme is responsible for filling in the gap left when the last RNA primer is removed? |
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Definition
| Telomerase (it is a type of reverse transcriptase) - it fills in the gaps as well as extends the length of the telomere |
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Term
| What are some interesting facts about telomerase? |
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Definition
| Activity and length decreases with age; primary cell culture lines have no telomerase activity; immortalized cell culture lines divide indefinitely b/c of activated telomerase; activated in cancer cells and believed to contribute to immortalization |
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Term
| Where is gene expression controlled? |
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Definition
| Pre-transcriptional, transcriptional, post-transcriptional, translational, and post-translational levels |
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Term
| DNA replication must be these three thing |
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Definition
• high fidelity (but need mutations)
• highly processive
• relatively fast |
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Term
| Semiconservative replication |
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Definition
• both strands get a new stand
• this is the correct method |
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Term
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Definition
| • both strands stay connected and a new one is created |
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Term
|
Definition
| • random pieces are kept and others are replaced |
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Term
| Meselson and Stahl experiment |
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Definition
• demonstradted semiconservative
• applied CsCl gradient ultracenterfugation of DNA labeled with Heavy Nitrogen (15N)
• cells moved to growth medium containing normal N (14N)
• DNA was isolated at different times
• pictures taken during centerfugation
• found old strand of heavy DNA with new strand of light DNA |
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Term
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Definition
both strands serve as templates in the same compass direction
one would do 3'®5' and hte other would do 5'®3' |
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Term
| semidiscontinuous replication |
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Definition
| same as continuous, but broken up into bursts on the same double strand |
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Term
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Definition
| fragmented on both parent strands |
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Term
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Definition
• proposed that DNA polymerase can make one strand of DNA continously 5'®3' (leading strand)
• but the other strand would be discontinous in 5'®3' (lagging strand) |
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Term
| Okazaki's model had two experimentally testable predictions |
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Definition
• if short pieces of DNA are synthesized on lagging strand, should be able to catch w/ radiolabeling
• if the enzyme DNA ligase is eliminated from the replication process, then short pieces of DNA made should be detectable even at longer labeling periods |
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Term
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Definition
• used T4 phage mutant that made Ligase
• control - found short pieces for short periods of time before they were ligased together
• using the T4 mutant - tons of short pieces as time increased |
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Term
| DNA replication is _____ with DNA synthesis occuring ____ at ____ _____ ____ |
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Definition
• bidirectional
• simultaneously
• 2 replication forks |
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Term
| specific location where replication starts |
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Definition
• oriC
• contains 4 9-mers having consensus sequence of TTATCCACA |
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Term
|
Definition
• an ATP-dependent enzyme that separates the DNA strands in advance of the replication fork
• the dnaB gene product in E.coli |
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Term
| Single-Stranded DNA-Binding Proteins |
|
Definition
• bind to ssDNA and prevent it from reforming dsDNA
• product of ssb gene of E.coli
• the bonding is cooperative - raises affinity by 1,000 fold for next molecule
• stimulate replication |
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Term
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Definition
• as the DNA unwinds, it has to wind somewhere upstream, creates strain
• topoisomerase releaves this strain by introducing temporary single- (Type I) or double-stranded (Type II) breaks in the DNA |
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Term
|
Definition
• from E. coli
• type II topoisomerase |
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Term
| DNA Polymerases found in E. coli |
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Definition
• Polymerase I - DNA repair, primer excision
• Polymerase II - SOS repair
• Polymerase III - required for DNA replication in E.coli |
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Term
| Holoenzyme polymerase III subunits (aka: pol III holoenzyme) |
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Definition
• composed of multiple subunits
• The α-subunit has the DNA polymerase activity
• The ε-subunit has the 3’ à 5’ exonuclease proofreading activity
• The θ-subunit has the ???? activity. The function is still unknown |
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Term
| eukaryotic DNA polymerases |
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Definition
• DNA polymerase α (priming)
• DNA polymerase δ (elongation)
• DNA polymerase β (repair)
• DNA polymerase ε (repair)
• DNA polymerase γ (mitochondrial) |
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Term
| DNA replication can be divided into 3 major events |
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Definition
| • initiation ® elongation ® termination |
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Term
| initation of replication - purpose of dnaA |
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Definition
• binds at the oriC
• facilitates the binding of dnaB
• stimulates melting of the 3 13-mer repeats at one end of the oriC to make an opening |
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Term
| initiation of replication - purpose of dnaB |
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Definition
• required for protein synthesis
• dnaC binds to dnaB
• stimulates binding of the primase
• also serves as the helicase that moves 5'®3' on the lagging strand in the direction of the replication fork |
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Term
| initiation of replication - two other factors for open complex formation at oriC |
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Definition
• RNA polymerase which synthesizes a short piece of RNA that creates an R loop
• helix unwinding (HU) protein which induces bending |
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Term
| Initiation of replication - primase |
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Definition
• product of the dnaG
• its the RNA primer-synthesizing enzyme |
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Term
| initiation of replication - Primase (dnaG) + dnaB = |
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Definition
• primosome
• this is responsible for laying down multiple primers for Okazaki fragments on the lagging strand |
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Term
| how eukaryotes handle initiation |
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Definition
| • multiple sites of replication for each chromosome |
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Term
| elongation - why pol III holoenzyme is highly processive |
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Definition
• due to the sliding clamp
• its the β-subunit of the holoenzyme
• literally holds the entire pol III assembly on the template for long periods |
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Term
| elongation - group of protein required for the sliding clamp |
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Definition
• the γ-complex
• sliding clamp cannot touch the DNA by itself
• serves as the clamp loader and is ATP-dependent |
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Term
| elongation - eukaryotes version of the sliding clamp |
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Definition
• processivity factor PCNA
• PCNA = proliferating cell nuclear antigen
• forms a trimer (3 subunits) that can encircle the DNA as the bacterial clamp does |
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Term
| elongation - pol III holoenzyme |
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Definition
• contains two core polymerases - one for each strand
• as it finishes one Okazaki fragment, it runs into a nick that is positioned in front of the primer on the next fragment
• this nick is a cue for the complex to dissociate from the and move to the primer on the next fragment |
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Term
| termination - the _____ region |
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Definition
• terminus
• where the replication forks begin to get near each other
• contains 22bp sites that bind specific proteins called TUS proteins
• replication forks stop moving when they get to this region
• leaves the daughter couples entangled |
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Term
|
Definition
| • terminus utilization substance |
|
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Term
| termination - when circular DNAs are interlocked (entangled), the structure is called ________ |
|
Definition
|
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Term
| termination - Decatenation |
|
Definition
• process of untangling the interlocking DNA rings
• performed by topoisomerase IV |
|
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Term
| eukaryotes' termination problems |
|
Definition
• there are gaps left when RNA primers are removed
• this is problematic because when the primer is removed DNA cannot be extended in the 3'-->5' direction
• and there's no 3' upstream
• so DNA should be getting shorter… |
|
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Term
| terminaton ends - which is which |
|
Definition
• TTGGGG - Tetrahymena
• TTAGGG - vertebrates |
|
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Term
|
Definition
• Hayflick (1960s) showed that normal animal cell lines are not immortal
• grow in cultures of about 50 generations, then enter senescence
• cancer cells do not have this limit (contain telomerase as do egg and sperm production cells) |
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