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Lecture 7

Lecture 7 8 Mitosis and Cell Cycle control .docx

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Western University
Biology 2382B
Robert Cumming

Lecture 7/8 – Mitosis and Cell Cycle control Function of the cell cycle  Essential mechanism by which all living things reproduce and pass on genetic information to next generation of cells o Replenishing cells  Ensures that DNA in each chromosome is faithfully replicated to produce 2 copies o To be a properly copy, have to be true to the original  Replicated chromosomes must be accurately distrusted (segregated) to 2 genetically identical daughter cells o To get two cells – you need to have the original cell grow a bit so that the daughter cells will be close to it in size rather than being scrawny things  Coordination of growth (increase in cell mass) with division The cell cycle and its phases  Mitosis is the most dramatic of the cell cycle  Cell cycle is the ordered sequence of events in which a cell duplicates its chromosomes and divides into two genetically identical  Four phases  G1-S-G2 = interphase, the cel increases in size  Most dramatic observed microscopally occur during the M-phase Phases of the Cell  Each phase has certain features  G1 (Gap1) phase o This is where most cells will be o Generalized growth and metabolism of the cell o Where most cells arrest when not dividing (G ) 0- When they’re completely out of the cell cycle  They’re on vacation, sometimes for all your life or sometimes short peiod of times o Variable length (~11 hours in mammalian cells)  S( Synthesis phase) o DNA Replication (~6-8 hours)  G2 (Gap2 Phase) o Preperation for chromosome segregation and cel division (~4 Hours)  M (mitotic Phase) o Chromatin Condensation  When moving DNa around you don’t want it to be all over the place, want it to be in a compact structure so you can move it around more easily  Making the DNa and the associated histones more compact o Nuclear envelope breakdown o Sister chromatids attached tomitotic spindles o Segregation of chromatids Lecture 7/8 – Mitosis and Cell Cycle control  Pulling them to separate poles o Decondense and reformationof intact nuclei o Cytokineasis (~1 Hour)  Pinching in half and dividing of the cell In cells that are in G1, while there is a large number of cells you have a lesser number of cells, as youre dividing and moving into G2M what’s important is the cells have completely replicated their DNA but have not yet dividied – that’s why you have almost DOUBLE the amount of DNA and thus DOUBLE the intensity (They have not yet divided) The peak of G1 is higher here beucase there tends to be the phase that cells are most likely to be in – the number of cells and the stain intensity has nothing to do with the stain intensity in the graph given on the powerpoint HOESCHST STAIN INTENSITY IS PORPORTIONAL TO THE AMOUNT OF DNA Some Eukaryotic Cell-Cycle Times  The complete process itself takes a while – but depends on the area and what cells you’re looking at  i.e. early frog embryo cells can do the full cell cycle in 30 minutes – that’s really fast!  Yeast cells in 1.5-3 hours which is relatively fast  Human liver cells – about 1 year , this is considered pretty long o So most human liver cells are in G1 or G0 phase  Human nerve cells (brain cells) – Terminally differentiated post mitotic cells o They’ve fallen into G0 and will stay there forever o They divided during embryogenesis and then they stopped dividing and that’s what you have for the rest of your life M-Phase (Mitosis)  Prophase o Nuclear envelope breaks down, spindle apparaturs forms, chromsomes condense o Early stage of mitosis where you break apart the nuclear membrane – the chromatin has begun to condense o You have the formation of the spindle poles and the spindle fibers – made of tubulin o These can elongagte and attach to the chromosome themselves (the centromeres)  Metaphase o Lining up the sister chromatid and attaching them  Anaphase o Sister chromatids separate pulled towards the spindle poles  Telophase Lecture 7/8 – Mitosis and Cell Cycle control o Chromosomes decondense reassembly of nuclear membranes o Reassemble the nuclear membrane – the spindle apparatus breaks apart o And then you get cytokensis – the cell splits apart  WANT TO MAKE SURE THAT THIS IS A CONTROLLED PROCESS  Otherwise you get ANIOPLOIDY – This is where you have wrong number of chromosomes in place to place  The only time you see cells dividing is really just mitosis Cytological features of cycling cultured human HeLa Cells  When cells round up it interferes with optical properties of cells thus you get the halo during phase contrast  Can’t see chromatin very well  HeLa continually cell cycling, it’s a bit unorganized but it does go through all the different stages  During G1 S G2 cells generally LOOK THE SAME TWO DIFF KINDS OF YESTCELLS The Budding yeast S.Cerevisiae  One of the fundamental properties of cell cycling was determined using yeast cells  It has what’s called a mother cell and a daughter cell  The mother cell will astart to form a bud – this usually happens during late G1 phase  This bud progressively increases in size as we move through the cell cycle  Cell cycle stage can be roughly inferred by the size of the bud  The bud is always much smaller than the mother cell  Once the daughter cell has finished will bud off and willg row to replae the original mother cell  G1 generally is the longest phase in these buding yeast cells Fission Yeast S. Pmbe  As it moves through the cell cycle it gets longer and longer - growing in length  Once it’s hit mitosis, unlike the budding there is no cytokenisis, there is a wall in the middle of the lage tube that forms (after DNA has been moved to each side )  G2 here tends to be longer  This is important because mayb e we’re more interested in G1 – than we’ll use the budding yeast  If we’re interested In looking G2 to M  These yeast cells are good because they’re easy to manipulate – easy to check the effect o mutations Lecture 7/8 – Mitosis and Cell Cycle control o In both SCerevisiae and S. Pombe temperature ssentive mutatnts exist which cause defects in specific proteins required to progress through the cell cycle o At certain temperatures the protein won’t work properly but as you decrease it the protein can fold properly and work properly o These mutants were very impmortant (CDC – cell division cycle mutatnts) were very important in figuring out features of the Cell cycle Concept of the Cell Cycle Control System  With the yeat cells determined that some of the major regulatrs are kinases – kinases are enzyems that stick phosphate groups onto amino acids o These are specific enzymes  Progression through cell cycle is regulated  System based on cyclically activated kinases  Checkpoints ensure things shouldproceed o Is all DNa replicated ? o Is the cell big enough? (During G2 has the cell grown big enough) o Is enviroment favorable? o Is DNA damaged?  Machinery stops if things go wrong o DNa replication is not perfect – errors happen, there are self correcting mechanisms and part of that Is stopping the Cells and fixing the problem as opposed to propagating it Involves 3 proteins families 2 of which are enzymes 1. Kinasese a. Put phosphate groups on 2. Phosphatases a. Take phosphate groups off 3. Cyclins a. Interact with the Kinases, act as regulators of the kinases Functional complementation  Temperature sensitive CDC mutants  If we take the take the cells and grow them at 25c they can form colonies o If you move them to 37c though it can not grow colonies  You can take advantage of this to take a plasma library – taking RNA from the wildtype normal cells, reverse transcribing it to make CDNA and putting all those CDNA into a Plasmid – you can then incorporate it into the temperature sensitive into the mutants  If it happens to take up a gene which takes the place of the mutant one the protein will now function at37c  This is to complement the deficiency  The growth cell division deficiency because we artificially the proper versio of the gene that will work at the nonpermissive temperature Lecture 7/8 – Mitosis and Cell Cycle control  Plasmid that carries WT allele will complement the recessive mutation  ARTIFICALLY INTORDUCING THE GENE THAT WORKS  There will be lots of genes (Gene x and Gene y) that won’t work!  But the few that take up the COMPLEMENTARY GENE it will work!  And you can isolate the plasmid of the colonies and figure it out  The original mutant is called cdc28
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