7Cells must be able to proliferate - during development- wound healing- stem cells in blood, small intestine, immune systemFor cells to copy themselves they need to:- Grow; make more stuff; e.g. proteins, lipids- Copy their genetic material- Segregate contents to daughter cells, especially…- Segregate replicated chromosomes to daughter cells
9Cell Cycle Commandments A cell resting in quiescence (G0) must not reenter the cycle unless a proper mitogenic signal is received.A terminally differentiated cell must not reenter the cycle.A cell must not start to replicate DNA unless its mass is sufficient to support cell division.A cell must replicate every DNA sequence once, and only once, during each cell division.If the DNA is damaged, a cell must repair the damage before cell division.A cell must not divide until DNA replication has been completed.Each cell must receive a complete complement of replicated DNA.
12Loss of cell cycle control at the Restriction Point Two types of genes are mutated in cancer:G0MG2SG1proto-oncogenesActivity: stimulate cell cycle progressionMutation in cancer: gain of functionproto-oncogene = wt; oncogene = mutantExamples: cyclin D1, Mdm2, myc, rastumor suppressorsActivity: Inhibit cell cycle progressionMutation in cancer: loss of functionExamples: Rb, p53, p16, ARF, PTEN
15S Phase of the Cell Cycle During the S phase, the duplicated DNA is rearranged through cohesion to form two sister-chromatids attached to each other by cohesinsThe cohesins will be removed during mitosis to allow sister-chromatid separation
23G1-S and G2-M are the major control points in the cell cycle Fuse M phase cell with interphase cell: Interphase nucleus enters MFuse S phase cell with G1 cell: The G1 nucleus enters S phaseFuse S phase cell with G2 cell: The G2 nucleus does not enter S phaseRao and Johnsoncell fusion experiments
24Cyclin Dependent Kinases Regulate the Cell Cycle
25Phosphorylation of CDK Targets Changes Their Activity Now performsa cell cycle function
26Experimental Systems Important for Cell Cycle Studies Saccharomyces cerevisiaeArbacia punctulataSchizosaccharomyces pombeXenopus laevis
28Budding Yeast: a genetic eukaryotic model organism Hartwell was interested in the protein synthesis machineryLee HartwellLet’s look for mutants that cannot synthesize proteins
29Isolating Temperature Sensitive Mutants in Haploid Yeast
30Serendipity: a scientist’s best friend! Lee HartwellBrian ReidBrian Reid, an undergrad, needs to look at a microscope to follow a mutant. They realize that bud size stores information about the cell cycle
31Cdc Mutants Arrest at the Same Cell Cycle Phase Permissive (low) temperatureRestrictive (high) temperature
32The Behavior of a Temperature Sensitive cdc Mutant cdc mutant growingat permissive temp (23C)cdc mutant growth arrested after6 hrs at non-permissive temp (36C)
38yeast are really cute and interesting, but This is all great andyeast are really cute and interesting, butCan we really learn something from all of this about humans?Schizosaccharomyces pombe
39Crazy ideaIt worked for us with budding yeast genes. Why not try human genes?Sir Paul NurseLet’s try to complement (rescue) the cdc2 (-) mutant of pombe with a human cDNA library
40Human cdc2 rescues cdc2 mutants!! Melanie Leecdc2 mutants, complemented by a human cdc2 geneElongated cdc2 mutants, failing to undergo mitosis
41Summary- A genetic approach in fission and budding yeasts reveals genes that are essential in promoting the cells through the cell cycleKey genes encode a protein kinase called CDKs for Cyclin-Dependent KinasesCDK1 = the protein encoded by cdc2/CDC28What about cyclins? How were they discovered?
42Woods Hole Marine Biological Laboratory Tim Hunt
43Cyclin was Discovered in Sea Urchin Embryos can stimulate tolay lots of eggsProteinLevelTimecyclin Acyclin BM
44(Spisula is actually a clam.) mitosis(Spisula is actually a clam.)
45OK, but what does this have to do with CDK’s?? I have theanswer!
46Overview of the frog life cycle OOCYTE GROWS WITHOUT DIVIDING(MONTHS)FERTILIZED EGG DIVIDES WITHOUT GROWING(HOURS)FERTILIZATION1 mmspermtadpole feeds, growsand becomes an adult frog