BIO 201 Cell Biology with Todd Hennesey Final Exam Study Guide
BIO 201 Cell Biology with Todd Hennesey Final Exam Study Guide BIO 201
Popular in CELL BIOLOGY
verified elite notetaker
Popular in Biology
This 96 page Study Guide was uploaded by ChiWai Fan on Wednesday May 4, 2016. The Study Guide belongs to BIO 201 at University at Buffalo taught by TODD HENNESSEY in Spring2015. Since its upload, it has received 274 views. For similar materials see CELL BIOLOGY in Biology at University at Buffalo.
Reviews for BIO 201 Cell Biology with Todd Hennesey Final Exam Study Guide
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 05/04/16
Cell Biology with Todd Hennessey—Study Guide for Final Exam: (images taken from Professor Hennessey’s slide; information edited by ChiWai Fan) Prokaryotic Cell Division (Bacterial Cell Division)—got one piece of closed circular DNA Make sure daughter DNA is identical to mother! One piece of closed circular double-stranded bacterial DNA Daughter cells (one to two) 3 What would happen if you added radioactive thymidine ( H-T) during DNA replication? Would both daughter cells be radioactive? YES. How is DNA replicated? Meselson-Stahl Experiments 1. Grow bacteria on “heavy” nitrogen ( N) to metabolically label all DNA (N-15 is radioactive nitrogen) creating all heavy nitrogen 2. Then switch to regular “light” nitrogen ( N). All newly replicated DNA will be “light”, but the old parental DNA will be “heavy” This way you can tell which DNA is new and which is old. You want to know how they replicate from parental cell to daughter cells. (Unnatural to natural) Anything light means they’re newly made How could you separate heavy DNA from light DNA? Density gradient centrifugation 3. Use density gradient centrifugation to separate heavy (higher density) and light DNA. They actually used cesium density gradients instead of sucrose. Separation of Bacterial DNA by density centrifugation Hybrid=one strand heavy with one strand light. Add mix of light, heavy, and hybrid DNA to a Cesium density gradient 15 What would you see if you added “normal” bacterial DNA that is not N? You will only see N-14. First generation: one becomes two Second generation: two becomes four Three Possible Models for DNA Replication—hypothesis testing Blue is old. Red is new 1. Semiconservative replication (hybrid): produces molecules with both old and new DNA, but each molecule would contain one complete old strand and one new one. 2. Conservative replication (light and heavy): preserves the original molecule and generate an entirely new molecule. (Copy machine) 3. Dispersive replication (hybrid): produces two molecules with old and new DNA interspersed along each strand; disperses and come back again Can’t tell if it is dispersive or semiconservative because both are hybrid. But you can tell it is or is not conservative because it is light and heavy. Semiconservative conservative dispersive Semi-conservative replication—how DNA replicates First generation: can only tell if it is conservative Second generation: two sets of DNA, one hybrid and the other all light; second generation tells if it is dispersive or semiconservative If you started the other way around with N-14 then switched to N-15. You will have heavy and hybrid instead of light and hybrid. DNA Replication in Eukaryotes-we have lots of pieces of linear DNA Use metabolically labeled DNA and look at mitotic chromosomes If radioactive makes something, the product will be radioactive: this is metabolically labeling 1. Must use mitotic cells to see compacted chromatids 2. Use BrdU (bromodeoxyuridine) to substitute for thymidine in DNA A:T A:BrdU (Use BrdU for T) newly made DNA will have BrdU instead of T 3. Thymidine stains dark. BrdU doesn’t. Thymidine DARK. BrdU DON’T. (Newly made DNA will not stain dark because they have BrdU) 4. All T is dark, all BrdU is light, hybrid is dark If both sides of X are light, then it is all BrdU. If one dark, one light, then it is hybrid. Note: BrdU will not go into RNA, we’re looking at mitotic chromosomes. BrdU will go in newly made DNA (NOT RNA!) In Eukaryotes Note: All T is dark, all BrdU is light, hybrid is dark WENT FROM HYBRID TO ALL OF SAME KIND (RED STRANDS) Parental DNA actually opens up and gets doubled In Second generation IF IT IS LIGHT, YOU KNOW IT’S ALL BRDU. Both strands are new Thymidine=dark MITOTIC CHROMOSOME IS MADE OF TWO CHROMATIDS (LIGHT ONE IS ONE CHROMATID (double stranded) AND DARK ONE IS ANOTHER ONE CHROMATID (double stranded)) ALL DNA RPLICATION IS SEMI-CONSERVATIVE DARK BECAUSE IT IS HYBRID You can’t tell if it is semi conservative in first generation. But you can tell during second generation. How does DNA replicate? DNA replicates to make DNA. DNA transcripts to make RNA How can you tell it is replication and not transcription? A is paired with T What if it was transcription? A would be paired with U <-not transcription because there’s A-T The Cell Cycle—Not about cell division. It is about nuclear division (dividing DNA) Not all cells divide: such as Red Blood Cells Different cells spend different amount of time in M-phase and Interphase. Not all cells spend the same amount of time in each phase Mitosis is not cell division. It is nuclear division. You have to divide DNA before the cells can pinch off (cytokinesis) and come apart for cell division Mother cells double her DNA, divide and want daughter cells to have identical gene information. M-phase: Mitosis & Cytokinesis Interphase: G1 & S-phase & G2 Chromatin, chromatids, chromosome: we have 23 chromosomes Compact the chromatin and pair the chromatids (left) A Disperse chromatin. A thread-like single piece of double-stranded DNA (linear) with associated proteins, mostly histones (Seen in all through Interphase); disperse so you can compact it in Mitosis (right) Mitotic Chromosome (paired compacted chromatids) there’s 46 of these in humans. (Seen in Mitosis only) compacting DNA makes it easier to separate. 1 piece of chromatin is a single piece of double stranded DNA Compacted and dispersed Chromatin Why have compacted chromatin in mitosis? It can’t be replicated or transcribed. So identical pieces of chromatin to be separated; so both daughters have 46 o No DNA replication or transcription in MITOSIS Why have dispersed chromatin in interphase? So it can be transcribed and replicated (sisters are held together) 1. In M phase cell, the DNA and proteins in each Mitotic Chromosome form highly compact structure 2. In an interphase nucleus, chromatins are threadlike structures dispersed throughout the nucleus 3. During the interphase, DNA is replaced. Only a tiny portion of one piece of chromatin of many is shown 4. At the end of S the chromatids are held together at the centromere In what phases of the cell cycle can you see mitotic chromosomes? MITOSIS 2 chromatids (double stranded and genetically identical) paired together=1 mitotic chromosome Cell Cycle M-Phase: Nuclear division (mitosis) and Cell division (cytokinesis) YOU DON’T WANT TO DIVIDE CELL UNTIL YOU HAVE ALL Interphase: G1, S and G2. Transcription and translation happen mostly in G1 and G2. DNA is replicated in S phase only and very little transcription or translation in S G1: cell grows and carries out normal metabolism; organelles duplicate S: DNA replication and chromosome duplication (preparation of mitosis) G2: cell grows and prepares for mitosis by doubling amount of DNA Cell Cycle Highpoints Chromatin is dispersed in interphase for transcription and replication DNA replication only happens in S phase. Histones and centrioles are replicated in S-phase too in preparation for mitosis. (ie: We want four to go in mitosis so we can divide into 2 each normal ones) Chromatin condenses into chromatids and forms mitotic chromosomes in mitosis. Since it is condensed, transcription and replication stop. Little or no translation either in mitosis. Centrioles and centrosomes in the cell cycle Centrioles replicate in S-phase The mitotic spindle contains two centrosomes. Each centrosome has two centrioles Cell Cycle Exceptions (some): not all cells go through same type of cell cycle 1. No G1 or G2. Most or all of interphase is S-phase. Cells divide a lot but don’t grow much. Much mitotic activity. Examples: Embryonic cells, spermatogonia, etc. 2. Stuck in G1 or G0. No division at all. Terminally differentiated cells such as some neurons, some muscle cells, RBCs, etc. (brain cells replicate like crazy) A cell not supposed to replicate like crazy starts to replicate like crazy= cancer 3. Inducible. Can progress from G1 to S if triggered to do so. For example, lymphocytes can be stimulated to proliferate by exposure to antigens. Liver cells regenerate after injury. Lizard tails can grow back after being cut off; Cancer cells; Stem cells Do soluble factors control the cell cycle? Assay: Fuse two cells together and see if the phase that one is in causes the other to enter that same phase Result: Fusion with a cell in S-phase stimulated the G1 cell to go into S-phase. How could you tell? Add radioactive thymidine, if you add that to G1 cell, the cell will not become radioactive. If you add it to S- phase cell, it will be radioactive because it is newly synthesized DNA. Then you fuse them together. You’d see radioactive DNA (by autoradiography) if H radioactive thymidine was added. How could you verify this? Microinjection 1. Take out a little of the cytoplasm of an S-phase cell 2. Microinject it into a G1 phase cell. Microinject water into a control G1 phase cell 3 3. Grow both cells in H thymidine, do autoradiography, see if they are radioactive. If the microinjected G1 cell is radioactive, it means it entered S phase (DNA replication) Can G2 cells be stimulated to replicate DNA again? No change seen in either cell. Conclusion: G2 cells don’t respond to S-phase factor (whatever it is) Can G2 cells be affected by M-phase (mitosis) factor? How can you tell if the G2 cell starts mitosis? From linear dispersed into condensed Result: The DNA in the G2 cell started to condense (compact), like they were starting mitosis. Also, G1 can be converted to M and S to M by “M-factor”. There are a lot of factors floating around Conclusion: Both S-phase and mitosis are controlled by soluble factors Fusion of G1 phase cell with a cell in mitosis G1 chromatin becomes compacted due to exposure to the cytoplasm of a mitotic cell Fusion of a G2 phase cell with a mitotic cell If you fuse G2 cell and mitotic cell, you can tell which are compacted and which are dispersed A soluble factor from the mitotic cell causes the G2 chromatin to condense How can you tell when a G2 cell is starting to go into mitosis, the chromatin is condensed What are these soluble factors? 1. A soluble factor that causes progression from G1 to S 2. Another (different?) soluble factor that causes progression from G2 to mitosis MPF and Cyclin; Yeast=eukaryotic; Yeast is doubling in mitosis; MPF: Maturation-promoting factor. Goes up during mitosis and goes down, this stimulate entry into mitosis, as seen by compaction of DNA. MPF activity is a protein kinase activity. From G2 into mitosis when the DNA starts to compact There are many protein kinase, they phosphorylate specific protein depending on cell function Kinases are enzymes that phosphorylate specific proteins Cyclin: Part of the MPF complex which regulates its activity; Cyclin levels goes up or down to signalize Need both MPF kinase and cyclin regulator to go into mitosis The MPF is not active until Cyclin binds to it Cdk is a kinase; Cdk Cyclin complex is starter Activated MPF phosphorylates a protein It is the phosphorylated protein that stimulates progression to the next cycle Cdk protein is always present but its active site is not exposed (until it gets conformational change by binding with Cyclin) Cyclin protein is made only at a certain point in the cell cycle 1. Cyclin binding changes Cdk, exposing its active site 2. A protein substrate and ATP bind to Cdk. The protein substrate is phosphorylated 3. The phosphorylated protein regulates the cell cycle. Each Cdk has specific protein targets Two cyclins in yeast, a model eukaryotic cell One cdc kinase but two different cyclins that can bind to it (the Cdc/Cdk Kinase is always there) G1 cyclin stimulates progression from G1 to S-phase Mitotic cyclin stimulates progression from G2 to mitosis Kinase is the same but different cyclin; you don’t want the signal to be always on or have two at the same time;Kinase has to be stimulated to do things Cell cycle regulation; Note: The Cdk is always there but it is not active until the cyclin appears This cyclin gene gets transcribed in G1; genes are there but some genes are not expressed until signaled Why is turned on and then turned off? It needs stimulant Is this a G1 cyclin or a mitotic cyclin? It is a G1 cyclin because it goes from G1 to S phase Cdk is present in Mitosis but without cyclin it is not active Cyclin synthesis begins during G1 and Cyclin bines to Cdk, which becomes active. Cyclin breaks down then Cdk is inactive in DNA synthesis (S phase) Cell Cycle Checkpoints: Cell cycle arrest (like a factory) 1. “Sensors” detect DNA damage or cellular abnormalities; like some mutation 2. The cell cycle is arrested, stalling progression to the next step; all along the way 3. During this delay, DNA damage is repaired or the cell defect is corrected CdK inhibitor proteins can act as “molecular brakes” to stop the cell cycle from progressing to the next step; until you fix the problem. What happens if the checkpoint isn’t working well? Mutation, wrong amount of DNA, dead cell. Specialized Inspectors (proteins/ complexes) can check the checkpoint, if a problem exists, you stop the whole thing, and all in a guy to try to fix it then proceed Inspectors everywhere An example of a checkpoint protein p27 is a CdK inhibitor, it binds to cyclin complex as cell cycle arrest. It must be removed before the cell can move into S phase. P27 starts cell cycle arrest by locking up cyclin so you can’t go into S phase What would happen if a cell was missing p27? You can get a spontaneous mutation (random at any time) and lose p27. A lot of times, p27 activation is false alarm Effects of a mutation in a checkpoint protein The brown mouse is a p27 knockout mouse. It has no p27. Therefore, there is no cell cycle arrest protein to pause progression into S phase. No p27 = More S-phase cells, more cell division, more cells, bigger mouse Higher cancer risk? Yes. If cell damage, there’s no p27 to fix it If defective or removal of p27, higher chance of cancer risk Mitosis; First a little bit about eukaryotic DNA Telomeres and Centromeres Telomeric and centromeric regions of DNA are replicated but not transcribed; Not all DNA is Transcribed; How DNA is Packed into a Mitotic Chromosome DNA wraps around histones to form nucleosomes; organizing and shorten DNA so you can split it in half easier; This compacts, loops and forms mitotic chromosomes with a packing ratio of about 1:10,000 The core Particle is an octamer of 8 histones Two domains (parts) to each histone in the core: 1. Hydrophobic: Responsible for histone assembly to make the core by hydrophobic aggregation 2. Hydrophilic: Positively charged, hydrophilic, faces the water. (Remember, DNA is negative at pH=7.0). DNA wraps around the outside of the core (chromatin) In general, eukaryotic DNA is either compacted (ex.: mitotic chromosome) or dispersed. Why? Dispersed chromatin is easier to replicate and transcribe. However, it is difficult to separate in mitosis Two pieces of compacted chromatin (mitotic chromosome) are easier to separate. However, it is more difficult to replicate and transcribe Condensin helps to compact and supercoil DNA in mitosis In which part of the cell cycle would you expect to see condensin first appear? M phase Condensin helps to compact DNA when starting mitosis. No condensin in interphase. Cohesin helps to keep the two pieces of chromatin together at the centromere after S-phase, through G2 and into the beginning of prophase Phosphorylation of cohesin in early prophase causes a loss of cohesin (after S phase) between homologous chromatids except near the centromere How could phosphorylation regulate this? Conformational change and change in charges by adding negative charge causing repel In which part of the cell cycle would you first see cohesin? S phase for DNA replication The kinetochore is a protein complex that is attached to the centromere o DNA is blue o Kinetochores are green; does not wrap all around the centromere; o Cohesin is red Cohesin holds the sister chromatids together at the centromere; the kinetochore is not always at the center of mitotic chromosomes Regulation of gene expression by histone modifications (FYI) Closed chromatin DNA inaccessible: transcription repressed Open chromatin DNA accessible: transcription activated Human karyotype: Not all centromeres are at the center Spectral karyotyping is a molecular cytogenetic technique used to simultaneously visualize all the pairs of mitotic chromosomes in an organism in different colors Mitotic Chromosomes are different in different cells. Different number of mitotic chromosomes, different sizes, different shapes DNA content and Ploidy Haploid: Single set of genetic information. Single genome 23-sex cells (1x) sex cells Diploid:Two full sets of genetic information. Two copies of the genome 46 (2x) Polyploid: Many sets of genetic information. Many genomic copies (up to 800 in cotton) Ploidy (n): Number of sets of genetic information. Number of copies of the genome. x: Mass of DNA or amount of DNA c: Number of base pairs (size of DNA) Human cells are mostly 2n2x2c in G1 In this course, the x and c numbers will always be the same Changes in human DNA content during the cell cycle In G1: 22 autosomes and 1 sex chromosome (XX female or XY male) That’s 23 kinds of disperse chromatin in G1. But there are two sets of each because we are diploid (2n) so: 46 pieces of disperse chromatin in G1. They are all linear, double-stranded DNA. In S: Each piece doubles so: 92 pieces of disperse chromatin in S-phase. The nucleus is still diploid (2n). Same thing in G2. In mitosis: The 92 pieces of disperse G2 chromatin condense in mitosis to form 92 chromatids. The 92 chromatids are paired to form 46 mitotic chromosomes. Still diploid (2n) DNA changes during the cell cycle For example, here’s a G1 NUCLEUS (human) right after fertilization S-phase The disperse chromatin doubles in S-phase There are 92 pieces of disperse chromatin in S-phase How many mitotic chromosomes in human S-phase cells? None; only mitotic chromosomes in M phase DNA ploidy and content during the cell cycle Goal of mitosis: Make two copies of cells with the same ploidy and DNA content as the mother. This is: Equational Division—no change in Ploidy (n) Mitosis: Mitosis is preparation for cytokinesis Phases of Mitosis Would you expect to see any cohesin connections in anaphase? No. Condensin? Yes 1. Condensin goes to work in prophase to compact DNA 2. Cohesin holds together the sister chromatids at their centromeres 3. Cohesin lets go in anaphase 4. Condensin starts to disappear in telophase and the chromatin becomes disperse Now back In G1 There’s both condensin and cohesin in Prophase Phases of Mitosis Prophase Chromatin cohesion in S-phase and compaction (condensing) in prophase of mitosis How can you tell you’re in prophase? 1. The chromatin is compacted. 2. Mitotic chromosomes are seen but they aren’t clustered near the middle 3. MTs from the mitotic spindle have not made contact with any mitotic chromosomes Prophase Prophase: Spindle formation 1. Pericentriolar material surrounding centrioles serves as the nucleation site (MTOC) for cytoplasmic microtubules 2. The initial spindle pole forms outside of the nucleus 3. Breakdown of the nuclear envelope, ER and golgi happens in prophase growing out towards plus end Not charges, they’re poles for plus end etc Prometaphase Chromosomal microtubules from the mitotic spindle begin to make contact with kinetochores Mitotic chromosomes begin to arrange in Amphitelic orientation Amphitelic means that each sister chromatid faces opposite poles Prometaphase Prometaphase 1. A protein complex on the centromere is called the kinetochore. 2. The plus ends of chromosomal MTs start to “capture” mitotic chromosomes at their kinetochores 3. Mitotic chromosomes begin to move (oscillate) and end up near the spindle equator at the end of Prometaphase 4. Sister chromatids end up in amphitelic orientation. Each faces opposite poles. In Prometaphase Three types of cytoplasmic MTs in the mitotic spindle: Not all cytoplasmic MT are the same 1. Astral MTs: Eminate from centrosome into cytoplasm to anchor and position the aster (mitotic spindle) 2. Chromosomal MTs: Connect from Pericentriolar material of centrosome to kinetochores 3. Polar MTs: Extend from centrosome to equator but interact with other polar MTs instead of chromosomes Three types of cytoplasmic MTs in the mitotic spindle: 1. Astral MTs: Eminate from centrosome into cytoplasm to anchor and position the aster (mitotic spindle) 2. Chromosomal MTs: Connect from pericentriolar material of centrosome to kinetochores 3. Polar MTs: Extend from centrosome to equator but interact with other polar MTs instead of chromosomes What’s the point: Cytoplasmic MT is dynamic and always growing and shrinking (Dynamic instability). Mitotic spindle can disappear in low temperature if they keep shrinking. Two of the main forces for movements in prometaphase You have mitotic chromosome, we want them to connect to MT and move around by dancing around until they find their position in the middle. 1. Polymerization (longer) and depolymerization (shorter) of tubulin to lengthen and shorten MTs. If you shorten the MT, it will pull towards pole of mitotic spindle. It heads away the pole by making MT longer. You do this to effect the movement. (like pull a rope with something attached) 2. “Cargo and rail” action of motor proteins on MTs to position mitotic chromosomes and poles. Dyneins and kinesins. 1. Tubulin polymerization and depolymerization and mitotic chromosome movements in Prometaphase You can only add to plus end to make longer and you can only make shorter by taking away from the minus end (Old thinking) now we know you can either subtract or add to each end. They’re growing and shrinking which provides force for movement (does not need ATP) it is not ATP dependent. We are using the dynamic instability (lengthen and shorten) for force of movement. What does this do? Lengthen and shorten chromosomal spindle fibers What would happen to the length if you stopped growth of these MTs? You can add inhibitor that inhibits MT polymerization, this will stop growth and cell will stop dividing. FYI: A depolymerase helps to shorten the chromosomal MTs at each end during prometaphase Can chromosomal MTs shorten at both the plus and minus ends? YES Dam ring prevents things from falling off. Growth polymerization at either end!! Not just plus end 2. Motor proteins position the mitotic chromosomes and the poles Which phase is this? Prometaphase: made connection and in the process of lining up towards the middle of mitotic spindle. We lost nuclear membrane when we came to prometaphase. Prophase: no connection with MT and mitotic chromosomes. So we are in prometaphase Remember, Kinesin is a plus-end directed motor. It moves the cargo towards the plus end 1. Polar spindle fiber MT sliding by Kinesin causes the poles to move apart; positioning the poles to make the mitotic spindle. 2. Dynein-based movement moves mitotic chromosomes toward the minus ends of chromosomal spindle fibers (going away from centrosome) 3. Kinesin-based movement moves mitotic chromosomes toward the plus ends of chromosomal spindle fibers. Chromosomal spindle fiber want to make contact with kinetochores, polar spindle fibers want to make contact with other polar spindle fibers. Chromosomal MT help position mitotic chromosomes; Polar spindle fiber help to position the poles (Polar spindle fiber can’t actually connect to kinetochores but chromosomal spindle fiber can!) Dynein goes towards centrosome. Kinesin goes away from centrosome All MT in mitotic spindle connect to a mitotic chromosome: FALSE!!! There’s other stuff. Metaphase Mitotic chromosomes align in Amphitelic orientation at the metaphase plate. Amphitelic means that each one of the two sister chromatids face opposite poles This is in the middle (equator) of the mitotic spindle. Colchicine and colcemid (inhibitors of MT polymerization) produces metaphase chromosomes because it stops them from being pulled apart in anaphase B. How can you tell you’re in metaphase? You look at it...above How many sister chromatids in metaphase of humans? 92! Is this a eukaryotic cell? Eukaryotic=true nucleus. NO we have no nucleus (DNA surrounding by membrane). Not all eukaryotic cell have nucleus. Spindle Checkpoint assures that all mitotic chromosomes reach the spindle equator before anaphase begins o Tubulin (green) o Mitotic Chromosomes (blue) o Spindle checkpoint protein (pink) o One mitotic chromosome that isn’t aligned has the spindle checkpoint protein (pink) bound to it. It starts cell cycle arrest. Remove checkpoint protein until pink has made it in. Have to wait for pink before we divide to prevent abnormal distribution of chromosomes. This won’t cause cancer but it may be a contributing factor, since this leads to genomic instability. “Inactivation of cell cycle checkpoints is a major cause of genomic instability and cancer in cells.” An improperly attached chromosome stalls the cell in metaphase a. No MT attaching to the right. Cell arrests at metaphase. (asymmetric tension triggers cell cycle arrest) b. Give tension applied with needle so the cell can proceed to anaphase What would happen if there was no checkpoint arrest? You can get abnormal number of chromosomes at both daughter cells Will this tricked cell produce normal daughters? NO Metaphase of Mitosis Paired homologous (identical) sister chromatids form 46 mitotic chromosomes Still diploid: 2n4x4c 46 pairs, all aligned at the middle of the cell Still 92 pieces of double stranded DNA (chromatin) but now they are compacted as 92 chromatids The paired 92 chromatids make 46 mitotic chromosomes. How do you know you’re in anaphase? Sister chromatids have come apart but they aren’t very close to the poles yet. Lose cohesin so you can split them apart but keep condensin. How many pieces of compacted chromatin in anaphase of humans? 92. How many cells in anaphase? One cell. Anaphase Homologous Sister chromatids separate Anaphase A. Chromatids move towards the poles by shortening the chromosomal MTs. shortening MT Anaphase B. Poles move apart. Slide spindle fibers to do that, cell will extend polar spindle fiber to push apart the poles. Sliding polar spindle fiber What causes the poles to move apart in Anaphase? A: shortening MT. B: sliding polar spindle fiber Anaphase A Poles don’t move. Individual chromatids move toward poles These sister chromatids have to be in Amphitelic orientation No change in the distance between the poles but MTs get shorter (the black lines are MT) What is the major force moving the sister chromatids in anaphase A? The shortening of MT due to dynamic instability. Does not require ATP. These are identical Anaphase B What is one of the major forces moving the poles apart in anaphase B? polar spindle fibers meet each other near the center with some kinesin in there. Now we have sliding pushing them apart. Movement of polar MT’s by MT sliding Telophase—still one cell! How do you know you’re in telophase? Chromatin becomes disperse and clustered near the poles. No mitotic chromosomes seen at the end of telophase. Starting to look like two cells but STILL ONE CELL that has two nuclei Cytokinesis 1. Contractile ring forms to pinch off cell at the center. This is microfilaments and myosin II 2. Two daughter cells produced, each in G1 3. Each cell has 46 strands of disperse chromatin. 4. Each is 2n2x2c. They are diploid all throughout Are both daughter cells genetically identical? YES Are they genetically identical to the mother cell? YES. Mom’s gone and now you’re the Mom Cytokinesis (the cell division)—obviously two cells Metaphase: 2n4x4c (92 chromatids, paired to make 46 mitotic chromosomes); Two daughter cells, both diploid (2n2x2c) Each daughter cell has the same 46 pieces of disperse chromatin in G1 after cytokinesis Identical daughters Cytokinesis Note: This is conventional myosin, myosin II What phase are the daughter cells in? G1 How many pieces of disperse chromatin in each daughter cell? After cytokinesis : 46 Identification of myosin II at the cleavage furrow with fluorescent antibodies Meiosis The goal of mitosis is equational division (for identical daughter cells) 2n2x2c 2n4x4c 2n2x2c MITOSIS The goal of meiosis is reduction division (2 ROUNDS) 2n4x4c 1n2x2c 1n1x1c MEIOSIS The goal of sexual reproduction is a return to the diploid state Mitosis is nuclear division (equational division). There is an S-phase in between the next mitosis (MITOSIS S-PHASE MITOSIS) Meiosis is 2 nuclear divisions with no S-phase in between (reduction division) reducing amount of DNA into just the necessary amount for an egg or sperm; you lose amount of DNA as you divide into sex cell Signals received in pre-meiotic cells trigger them (in G1) to start pre-meiotic S-phase. In other words, the decision about whether to go into mitosis or meiosis is made in G1. Body cell goes through only Mitosis. Sex cell goes through Mitosis and Meiosis. Primary meiocytes are cells that have just come out of pre-meiotic G2 and have entered prophase I of meiosis (first round) Meiotic prophase nuclei differ from mitotic prophase nuclei due to formation of synaptonemal complexes and bivalents (tetrads) in meiotic prophase I Only see bivalents tetrads in meiosis I. so you can use that to tell if you’re in meiosis 1 or meiosis 2. Bivalents (tetrads) in meiosis: o It is bivalent because it is two meiotic chromosomes o It is a tetrad because it has four chromatids o It is syntelic because both sister chromatids face the same pole In mitosis, copies of each chromatid align at the metaphase plate In meiosis, copies of each meiotic chromosome align at the metaphase plate (lining up mom dad) when split, they’re splitting differently and unevenly every time you shuffle the aligning Human karyotype o One mitotic chromosome. o Two pieces of condensed chromatin. o Two sister chromatids There are two identical double stranded chromatids above. In meiosis, you have two of these sets. o There are 23 bivalents in meiosis I o 23 pairs in metaphase of meiosis I First meiotic division (Meiosis I): we have twice as much DNA as in G1; Meiosis I produces two products Homologous chromosomes pair as bivalents or tetrads (2n4x4c) Intact meiotic chromosomes (as paired chromatids) separate No S-phase in between Second meiotic division (Meiosis II): no more bivalents; Meiosis II produces 4 haploid gametes Homologous chromatids separate; Each nucleus gets one set of chromatids Four haploid gametes, all 1n1x1c Mitotic Chromosome Paired sister chromatids Both chromatids are genetically identical Same thing in meiosis but we’ll call it a meiotic chromosome for clarity P2 paired together then separate when in meiosis 2 Bivalent (tetrad) in Meiosis I Homologous Chromosomes (homologous meiotic chromosomes) Paired meiotic chromosomes Both meiotic chromosomes carry similar information but they are not genetically identical What do you get when you pull them apart? Mitosis: you get chromatids. Meiosis 1: stay together. Meiosis 2: looks like mitosis, chromatids will come apart. Meiosis I and II The goal of Meiosis is haploidization Each gamete contains 23 chromatids (22 autosomes and 1 sex chromosome). Each gamete is 1n1x1c Cohesin in mitosis and meiosis Removing cohesin causes separation of sister chromatids in both mitosis and in meiosis II The sister chromatids are genetically identical They are held together with cohesin In metaphase, cohesin is only at the centromere Cohesin is removed at the end of metaphase in response to APC (anaphase promoting complex) This allows sister chromatids to separate in anaphase 1. During prophase, after DNA replication, the sister chromatids are held together by cohesin 2. In prometaphase, most of the cohesion is removed, except for some at the centromere 3. At the end of metaphase, a cyclin-Cdk complex activates the anaphase-promoting complex (APC), which activates separase, resulting in the removal of the remaining cohesin How does APC work? It acts by turning off existing signals o It is a multisubunit enzyme complex o It causes ubiquitination of many substrates. A substrate is what an enzyme acts upon. Ubiquitination attaches ubiquitin to a substrate. o Ubiquitination labels substrates for destruction. o A substrate with ubiquitin on it is targeted for degradation. (breakdown) o APC itself doesn’t destroy proteins themselves; it targets the proteins for destruction by other processes Ubiquitination targets proteins for destruction 1. A protein is targeted for degradation (breakdown) 2. An enzyme attaches ubiquitin to the protein and its gone 3. And the target protein is recognized by a proteasome (only to destroy proteins with ubiquitin on it) so you can turn over signals because you don’t want to have 30 signals to tell you to do things all at ones. You want one at a time and get rid of it afterwards. 4. Ubiquitin is released and recycled 5. The proteasome hydrolyzes the target protein Blue: proteasome What’s the point? The cell cycle can be regulated not only by the appearance of controlling factors, but also by their regulated disappearance Two ways to decrease protein amounts: decrease synthesis or increase degradation APC can specifically target proteins for degradation by ubiquitination Cohesin holds sister chromatids together: In mitosis (until anaphase) In meiosis I (until anaphase II of meiosis II) You should see APC in meiosis II only Two kinds of genetic variability in meiosis: doesn’t cause changes in DNA sequence & genetic mutations 1. Genetic recombination by crossovers 2. Independent assortment of maternal and paternal genes A mutation is a change in the DNA sequence of a gene A gene is a locus or region of DNA that encodes a functional RNA or protein product, and is the molecular unit of heredity Does all of DNA contain genes? NO Cross-overs and genetic recombination: All of the next 7 slides are in prophase I of Meiosis I Synaptonemal complexes in prophase I of meiosis I Homologous chromosomes are held together by synaptonemal complexes Cohesin holds together sister chromatids in each meiotic chromosome Synaptonemal complexes hold together bivalents Tetrad Recombination This happens in prophase I of meiosis Crossing-over and recombination occurs between (not within) non-sister chromatids (between red and grey; mom and dad) Would this cause a mutation? NO, this is good for evolution This is not recognized as a replication error and no checkpoint arrest is made Would a cross-over between sister chromatids change anything? It wouldn’t do anything. Because they’re genetically identical Extended meiotic prophase I 1. In the beginning of meiotic prophase I, chromatin condenses (DNA compacts) lots of condensin 2. Then homologous chromosomes pair (to form bivalents or tetrads) and synaptonemal complexes form (mom and dad pair up) 3. Crossing over and genetic recombination can now happen while the synaptonemal complex Is there 4. Near the end, synaptonemal complexes disappear and recombination stops. Now cross-over points hold them together The appearance of synaptonemal complexes is transient. They’re gone by the end of meiotic prophase I. How can you tell that this isn’t prophase of mitosis? Never see tetrads and bivalents, synaptonemal complexes, genetically mixing up in Prophase of mitosis. ONLY FOR PROPHASE I OF MEIOSIS Early prophase I Compacted meiotic chromosomes become visible Each is a pair of identical chromatids (from S phase duplication) How can you tell that this is meiotic prophase I and not meiotic prophase II? Twice as much DNA in Prophase I than Prophase II. Middle of prophase I A process called synapsis starts to pair homologous chromosomes Synapsis makes synaptonemal complexes Homologous chromosomes are held together by synaptonemal complexes (do recombination) Paired homologous chromosomes are called bivalents or tetrads There are 23 bivalents When the synaptonemal complexes are gone, chiasmata continue hold them together Now we leave prophase I of meiosis I Metaphase I of meiosis I This is still the primary meiocyte There are still 23 bivalents Is cohesin still there? YES Anaphase I of meiosis I Bivalents are gone. No more bivalents or tetrads in the rest of meiosis Paired sister chromatids begin to move towards the poles Telophase I of meiosis I This is still the primary meiocyte The first nuclear division is complete at the end of telophase I Cytokinesis follows to generate the two secondary meiocytes Meiosis II: contains half the stuff as meiosis I Prophase II: Since the chromatin was dispersed in telophase I, it must re-condense for prophase II for easy dividing The secondary meiocyte does not have bivalents. They are paired sister chromatids (like in mitosis) No genetic recombination in the secondary meiocyte. Why? We already did that in Meiosis I Metaphase II: Metaphase II has 23 pairs of sister chromatids at the metaphase plate. How many were there in metaphase I? 23 Bivalents. 46 pairs of sister chromatids. From 46 in Metaphase I to 23 in Metaphase II. Anaphase II: Sister chromatids separate. 23 pieces of compacted chromatin (chromatids) go towards each pole MEIOSIS I: When to hold and when to let go? 1. What holds the sister chromatids together throughout meoitic prophase I? o Cohesin When does all of the cohesin let go? Not until anaphase II of meiosis II 2. What holds the homologous meiotic chromosomes together in prophase I of meiosis I? o Synaptonemal complex in the middle of prophase I o Chiasmata in later prophase I When do these let go? In anaphase I 3. Why have synaptonemal complexes? In anaphase I o For Genetic Recombination (to add genetic diversity) good for evolution Which of the following are seen in both Prophase I and Prophase II of meiosis? A. Synaptonemal complexes-- NO, only in Prophase I B. Tetrads (bivalents)-- NO, only in Prophase I C. Chiasmata and genetic recombination—NO, only in Prophase I D. Copies of the entire maternal and paternal genomes—NO, only primary meiocyte does E. Compacted chromatin—YES Independent assortment of maternal and paternal genes Independent assortment after one complete meiosis: mixed up contribution from mom & dad Four gametes Independent assortment after a different complete meiosis Each round of meiosis can produce different grouping in each gametes Analogy for Independent Assortment: You have two decks of cards: • A blue deck. This deck has 46 cards and they are all blue on the back but there are 46 kinds • A red deck. This deck also has 46 cards but they are all red. They have similar 46 kinds, just different printing Mix them together on the floor. There are now 92 cards. Pull out 46 with your eyes closed. What did you get? Now put them back, remix and take out 46 again. Will it be a different mix? It may. Now add the effects of genetic recombination and independent assortment together The DNA sequences of each chromatid may be different. Each gamete can get different genetic information. Two sources of genetic variability in “normal” Meiosis: 1. Crossing over and recombination. This happens in prophase I It changes the distribution of maternal and paternal genes in some chromatids 2. Independent Assortment of genetic traits (maternal and paternal) This happens in Anaphase I of meiosis What does all of this do? It changes the distribution of maternal and paternal genes in each gamete. All of this contributes to genetic diversity There are two general kinds of cells in the human body: 1. Somatic cells. “Body” cells. Mitosis but no meiosis. Genetic mutations in somatic cells are not passed along to the progeny 2. Sex cells. Cells that differentiate into either egg or sperm cells. Can do mitosis or meiosis Only sex cell mutations are inherited Sperm and egg both doing mitosis but end differently: Differentiation Sperm growth then differentiation at the end (Meiosis Imeiosis IIdifferentiation) Egg growth and differentiation happen first (DifferentiationMeiosis IMeiosis II) Summary of Mitosis and Meiosis Mitosis produces daughter cells with the same genotype as the mother cell No genetic segregation or independent assortment in mitosis Meiosis produces cells with different mixtures of maternal and paternal genes because of: Independent assortment Genetic recombination Independent assortment and recombination happen in meiosis, not mitosis Goal of Mitosis: Faithful division of DNA so the daughter cells are genetically identical to the mother cell. Keep it real, keep it diploid, and keep it 100%. Goal of Meiosis: Mix it up. Produce haploid gametes with different genetic contributions from maternal and paternal sources for further genetic mixing by sexual reproduction. When things go wrong Aneuploidy. Abnormal number of chromosomes Recombination and Independent Assortment are supposed to happen. Aneuploidy shouldn’t Aneuploidy is when things go wrong. Recombination and independent assortment shouldn’t cause aneuploidy Aneuploidy can result from either primary or secondary nondisjunction Mistake happens in different stage Nullisomy is missing chromosomes. This is usually fatal Too many or not enough chromosomes can be bad Trisomy 21 (Down’s syndrome): example of aneuploidy Cell choices when DNA is altered by damage, mutation or mistakes: 1. Correct it. Arrest the cell cycle and clean up the mess before proceeding. 2. Evolve. Live with the change as a good mutation and as a selective advantage 3. Die (necrosis) because there is no choice: cells naturally die 4. Cell suicide (apoptosis): cell chooses to die before it gets mutated to the point of causing cancer 5. Cancer DNA Repair DNA damage and replication mistakes and happen. What to do about it? Repair the mistakes Some examples of DNA repair mechanisms: 1. Proofreading by DNA polymerase I 2. Nucleotide excision repair 3. Base excision repair 4. Double-strand break repair What happens if you can’t repair the mistake? Either cell death, suicide (apoptosis) or the DNA mutations can be passed on during replication. A. Germ cell (sex cell): Genetically inherited mutation B. Somatic cell: Cancer A pyrimidine dimer that has formed within a DNA duplex following UV irradiation This is an example of the kind of DNA damage your skin gets in the sun This can also be caused by Ionizing radiation, common chemicals and thermal energy Luckily, our cells have a number of mechanisms to repair this kind of genetic damage Nucleotide excision repair after UV damage Nucleotide excision repair (NER) removes bulky lesions, such as thymine dimers and chemically altered nucleotides. 1. DNA damage is recognized by a protein complex 2. The helicase activity unwinds the DNA 3. It cuts it 4. It removes the damaged sequence 5. The gap is filled by a DNA polymerase 6. The DNA is rejoined by a ligase If all these worked to repair, you are very unlikely to get cancer At least 4 different gene products (enzymes) are necessary for this Xeroderma pigmentosum (XP) Xeroderma pigmentosum (XP) patients cannot repair sun-damaged DNA via Nucleotide Excision Repair If tumor suppressor genes (p53) or proto oncogenes are also affected, the result may be cancer. Patients with XP are at a high risk for developing skin cancers. Apoptosis: Programmed Cell Death and Cell Suicide Why have apoptosis? Cell necrosis is ugly and messy. It generates all sorts of garbage; Apoptosis is a neat and orderly way to kill cells Apoptosis and removal of the dead cell by a macrophage Apoptosis generates caspases which are specific proteases. Examples of the target proteins for these caspases are protein kinases, nuclear lamins and cytoskeletal proteins Caspases can be activated to cause apoptosis by either an extrinsic pathway (stimulated by receptors) or an intrinsic pathway Programmed cell death during development The paw of this mouse embryo is being sculpted by cell removal. The yellow dots are dying cells stained for apoptosis Cancer There are many kinds of cancer Oncogenes and tumor suppressors Oncogenes are positive regulatory factors which have mutated to become overactive For example, HER2 is a growth factor receptor. Overproduction causes more growth stimulation Tumor suppressors restrict growth. If mutated or inactivated, growth increases Cancer Treatment and the Cell Cycle If cell growth is out of control, just STOP IT. Then fix it and clean up the mess. Now you can resume. You stop everything (good and bad) Restriction point (checkpoint): some drugs such as Herceptin, inhibit growth factor simulation at the restriction point Radiation damages DNA and causes apoptosis at the S and G2 checkpoints Effects of DNA damage 1. DNA gets damaged by radiation, mutagens, etc. 2. BRCA enzymes help repair the damage. If they are mutated or missing, damage persists 3. A checkpoint should catch this damage and activate p53 4. p53 is a transcription factor that triggers either: p21 expression and cell cycle arrest or apoptosis If no p53 and no cell arrest, the cells proceed to a tumor, cancer or death by necrosis p53 p53 (also known as protein 53 or tumor protein 53), is a tumor suppressor protein. It regulates the cell cycle and is involved in preventing cancers. P53 put cell cycle into arrest or tell cell to kill itself p53 has been described as "the guardian of the genome” because of its role in conserving stability by preventing genomic mutation. About 50% of human cancers have altered p53 p53 is a transcription factor that binds to DNA Specificity for binding is based on amino acid R is arginine, it has a charge of +1 at pH=7.0 An example of a p53 effect Detect damage of DNAactivation of p53 (p53 only do things when it is phosphorylated) In absence of DNA damage, p53 is degraded in proteasomes P53 is a transcription activator for p21. You only get p21 when DNA damage activates p53 which transcribes for p21. P21 now start cell cycle arrest. What would happen if the p21 gene was mutated? It does not cause cancer. It takes away cancer prevention. Which means higher risks for cancer What would happen if the damage can’t be fixed? Apoptosis, if p53 and p21 worked but still can’t fix then apoptosis. If apoptosis don’t work, then may lead to cancer. In general, most cancers are due to loss of cell growth control because of mutations in: A. Oncogenes: They ignore inhibitory signals (like checkpoints). For example about 50% of human cancers have altered p53. B. Tumor suppressor genes. They don’t need stimulatory signals for cell division if mutated “Cancer is a genetic disease… but in most cases, it is not an inherited disease”. What does this mean? Cancer is usually due to somatic genetic mutations that are not heritable. (You may inherit a genetic predisposition for it though.) Some ways that contributes to triggering cancer: 1. Mutate BRCA enzymes. DNA damage not repaired properly 2. Mutate p53. Less expression of p21 and loss of checkpoint control 3. Mutation in p21. Inability to respond to p53 and loss of checkpoint control BRCA, p53 and p21 are tumor suppressor genes Mutations can be either inherited or new. Mutation can either increase or decrease activities There are lots of different kinds of mutation. In general, cancer cells have problems with (all of these things): 1. Growth signals: A. Ignore inhibitory growth signals (tumor suppressors). B. Continue to grow in the absence of growth factors (oncogenes). 2. Defects in mitotic checkpoint proteins 3. Eliciting an apoptotic response Many cancers require multiple mutations in the same cell line Would a RAS gene mutation always cause cancer? Not always. Many mutations might cause problem. Probability of cancer might increase if you have defected cell already plus mutation. Clonal growth of a cancer cell Tumor growth often requires many somatic mutations in cells from a common origin All of these cells came from mitosis of the first cell in this line. Are they all genetically identical? Very low possibility to mutate an already-mutated cell. Mutation upon mutation is rare. Properties of cancer cells: Cancer cells are immortalized Cancer cells do not form differentiated tissues Cancer cells are not under contact inhibition Cancer cells are invasive (takes up other body part’s growing space; ex. huge liver) Cancer cells escape apoptosis An immortalized cell line Stem cells can differentiate, Cancer cells don’t, (cancer cells stay as cancer cells) Cancer cells are immortalized, just like stem cells, but without control Ex: Hela Cell Most normal cells stop proliferating under contact inhibition Loss of contact inhibition: cancer cells spread nonstop Molecular changes required for metastasis: Cells need to lose cell-cell adhesion contacts Cells need to penetrate through the matrix (Extracellular matrix help prevent cancer cell metastasis) Loss of Cadherins is detected in all tumors Metastasis A benign tumor is a mass of cells (tumor) that lacks the ability to invade neighboring tissue or metastasize. It is not spreading into other tissues but still grows. It is continues to get bigger, it can turn into malignant. (It’s better to catch it before benign turn into malignant) A malignant tumor has metastasized and spread out to other tissues What causes cancer? accumulation of Mutations Random mutations (mistakes at the assembly line) Inherited mutations (pre-disposition) Viral infections Environmental factors (chemical; physical) Mutations are heritable changes in the DNA sequence Genetic mutations are changes in the DNA sequence of genes (not all DNA carries genetic information) A gene is a locatable region of genomic sequence, corresponding to a unit of inheritance, which is associated with regulatory regions, transcribed regions, and/or other functional sequence regions Genotype: the DNA sequence Phenotype: the physical trait DNA: G-C ; A-T RNA: G-C ; A-U Codon usage table--Why no T’s in this table? Because it’s RNA. Codons are on mRNA Not all mutations cause changes in amino sequence. Not all mutations are bad. A point mutation changes only one of the nucleotides Do all point mutations cause changes in amino acid sequences? NO An example of the effects of point mutations 1. Since the mutant can’t make histidine, it can only grow if histidine is added, this is a His- mutant. 9 2. The rate of spontaneous mutations is about 1 in 10 nucleotides copied 3. If you screen enough of these bacteria, you can get a revertant that restores normal function What’s the point of point mutations? Point mutations can be either bad, good or do nothing One way for a mitogen pathway to affect cancer Mitogen tells cell to start growing G1-Cdk and G1/S-Cdk are kinases that trigger progression into S-phase Activation of these kinases causes phosphorylation of a protein that keeps a transcription regulator inactivated (Green thing is transcription regulator, it wants to tell the cell to grow; the red thing wrapped around green aka mitogen is trying to tell it to stay inactivated and do nothing) Genes get transcribed that produce proteins to stimulate cell division What could happen if Rb was mutated and inactivated? Now we don’t have transcription regulator. Now we have uncontrolled cell growth. So we hope checkpoints or apoptosis help the cell. If not due to many others having mutations too, then cancer. If you have cancer that means many regions of the cell have problems. Telomeres and Telomerase In many cells, the DNA gets shorter every time it is replicated. That may be one reason why we have long, untranscribed DNA (telomeres) at the ends. Telomeres are replicated but are not transcipted, they don’t code for any information In stem cells, they have an enzyme called telomerase, which puts on telomeres. Stems cells would have a problem if each time they replicate DNA get shorter because stem cells replicate a lot. The gene for telomerase is in all of our cells, but not all cells express telomerase. Telomerase makes sure you don’t lose DNA information when it shortens. Telomeres and cancer It is estimated that human telomeres can be up to 1kb long. They can lose about 100 base pairs from their Telomeric DNA at each mitosis. This represents about 16 TTAGGG repeats. At this rate, after 125 mitotic divisions, the telomeres would be completely gone IN SOME CELLS. Telomeres shorten as you get older When the telomeres are gone, there are problems with DNA replication and shortening of DNA strands. Cells deal with this by: o Ceasing to divide (replicative senescence) cells just stops dividing o Checkpoint arrest and DNA repair o Expressing telomerase to extend telomere length (cancer express telomerase and extend telomere so they never stop dividing) o Apoptosis, cancer or cell death Telomerase isn’t expressed in most mature somatic cells. Telomerase continues to be expressed in: o Germ cells; sex cells o Stem cells; as a baby so they can replicate a lot o Some protozoans like Tetrahymena (immortal) and keeps dividing o Cancer cells. Telomere elongation is often used as a diagnostic for cancer cells Telomerase Telomerase makes telomeres. It is an RNA-dependent DNA polymerase (it makes DNA from RNA) Telomerase was first discovered in Tetrahymena; (immortal, continue to divide as long as there is food) How does telomerase work? Telomerase, also called terminal transferase, is a ribonucleoprotein that adds a species-dependent telomere repeat sequence to the 3' end of telomeres 1. Telomerase binds to the 3’ end of DNA 2. The RNA template on the telomerase aligns with the DNA 3. Reverse transcriptase uses the open RNA sequences as a template to extend the DNA strand What is the genetic information in cells? The Griffith Experiment (1928*) 1. Two different strains of pneumococcus bacteria were used to infect mice with pueumonia. A. Rough strain. Doesn’t kill mice B. Smooth strain. Kills mice 2. Can the non-killer strain (rough) be converted into killers (smooth) by genetic transformation? What Griffith already knew? I. The phenotype is the observable properties of a cell or organism that are genetically controlled. A. Rough strains formed small, irregular colonies on nutrient plates that were easily recognizable. They are non-virulent (don’t kill mice). This is their phenotype. B. Smooth strains form indicative large, smooth colonies. Smooth strains of these bacteria are encapsulated and virulent (killers). This is a different phenotype. The capsule may prevent them from being killed by the mouse, allowing them to live and infect the mouse with pneumonia Hypothesis to be tested: The genotype of a cell is determined by its DNA. DNA contains the genetic information that is passed on from cell to cell during cell division. Genotype (DNA) determines phenotype. Griffith never answered his hypothesis Bacterial polysaccharide capsule Results of the Griffith Experiment 1. Smooth kills. These bacteria can live inside mice lungs because capsule protects bacteria and cause pneumonia to kill mice if there’s MANY bacteria replicated 2. Rough don’t kill. No pneumonia. Mice live. 3. Dead smooth strains don’t kill mice. To cause pneumonia, the bacteria must be alive so that the bacteria can replicate and cause the infection. They heated and killed Smooth so they can’t replicate thus didn’t kill mice. 4. Transformation of live Rough cells turns them into killers. Add dead smooth cells with something from rough, to turn them into smooth killers. What is the transformation factor in smooth cells that makes them killers? But Griffith didn’t figure out the transformational factor. What we know now that Griffith didn’t know Bacterial Transformation with foreign DNA (This changes the genotype of a cell) Little piece of foreign DNA got into genome of recipient cell and starts to replicate and express the foreign gene. Summary so far: 1. Rough strain pneumococcus does not kill mice 2. Smooth strain pneumococcus does kill mice by causing pneumonia Hypothesis: The genotype of a cell is determined by its DNA. DNA contains the genetic information that is passed from cell to cell during cell division. Genotype determines phenotype. Question to be asked: Can the phenotype of the rough strain be transformed by exposure to an extract of smooth strain cells so that they become killers? Did Griffith know what the transformation factor was? NO What was the Transformation factor? 1. It is heat stabile (not heat labile). Heating kills the bacteria but it doesn’t inactivate the transformation factor. 2. It must be incorporated into living bacterial cells. It can’t kill by itself. 3. What is the transformation factor? Is it protein, lipid, DNA, RNA or what? How could you tell? Would you expect the purified transformation factor to be toxic? No Avery experiments (1940) Test extracts of heat-killed smooth cells by treating them with either protease, RNAse or DNAse and injects it into mice with live rough cells A. Protease destroys proteins. Does it destroy the transformation factor? B. RNAse destroys RNA (but not DNA). Does it destroy the transformation factor? C. DNAse destroys DNA (but not RNA). Does it destroy the transformation factor? How can you tell if the transformation factor was destroyed? The mice live after being injected with rough cells and an extract that was treated with something that destroyed the transformation factor. What Avery saw? Took smooth bacterial, heat and filtered dead bodies and called that the extract and divided into 3 tubes. 1. Protease didn’t destroy transformation factor 2. RNAse didn’t destroy transformation factor 3. DNAse destroyed transformation factor so mice live. DNAse destroyed the transformation factor so it was DNA. Avery’s Conclusion. Not everyone buy it. Hershey-Chase Experiment (1952) Main Questions Asked: When a virus (phage) infects a bacterium and replicates, is the infecting agent DNA or protein? Is DNA the genetic information that is passed from the infecting virus to all of the progeny (new phages)? How some viruses can replicate: 1. Virus injects its DNA into a cell 2. Some of the viral DNA is replicated 3. Some of the viral DNA is transcribed 4. The transcribed RNA is translate
Are you sure you want to buy this material for
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'