New Material DNA Replica on Cell Cycle Cancer Cell Cycle I Cell Growth Division Cell Growth Division Overview division highly regulated embryonic development of cells size of organs ssues maintenance of form func on in adult wound healing regenera on immune response cell doubling requires doubling of all parts maintain cell size shape propor ons biosynthesis of all organelles regulated mechanisms conserved yeast worms frogs ies humans have conserved cell cycle machinery control decision to begin DNA rep or enter mitosis Cell Cycle Stages Cell Cycle Stages G1 8 10 hrs S 6 7 hrs G2 3 5 hrs G1 8 10 hr S 6 7 hr M 1 hr G2 3 5 hr M 1 hr G1 G2 essen ally Times are variable G1 G2 are essentially absent In early development absent in early development S M divisions S and M Divisions Methods to Study Cell Cycle Study Methods slide 1 of 2 Methods to Study Cell Cycle instrumenta on techniques mito0c index frac on of cells Instrumentation and Techniques in M phase Mitotic Index fraction of cells in M stain cells count those w BrdU Labeling Index Fraction of Cells in S Fluorescence Activated Cell Sorter Synchronization Methods nucleus frac on of cells in M represents frac on of me within full cell cycle spent in M random sample of cells BrdU labeling index frac on of cells in S phase Mitotic Shake Off all M phase Double thymidine block arrest in S Colchicine all M phase measure presence of T in DNA b c T is only found in DNA not mRNA measures S BrdU incorporates as T can be seen in microscopic stain Approaches and Biological Systems Genetics Yeast cdc Mutants Biochemistry Frog Egg Maturation sorter records light scawering Developmental Biology Sea urchin Surf Clam of each cell uorescence ac0vated cell incorporate BrdU measures S can see how many cells uorescence ac vated cell sorter Cell cycle control Methods to Study Cell Cycle Instrumentation and Techniques Mitotic Index fraction of cells in M BrdU Labeling Index Fraction of Cells in S Fluorescence Activated Cell Sorter Synchronization Methods Mitotic Shake Off all M phase Double thymidine block arrest in S Colchicine all M phase Approaches and Biological Systems Genetics Yeast cdc Mutants instrumenta on techniques Biochemistry Frog Egg Maturation mito0c index frac on of cells Developmental Biology Sea urchin Surf Clam in M phase Cell Cycle Study Methods slide 2 of 2 synchroniza on methods mito0c shakeHo all M phase double thymine block arrest in S phase add T block cells in S get stuck in S cells in G2 or later go around then get stuck in S use T block again before star ng cell cycle colchicine all M phase binds MTs prevents MT growth approaches biological systems gene cs yeast CDC mutants biochemistry frog egg matura on developmental biology sea urchin sea clam Cell cycle control ow cytometer measures amount of DNA present in a cell Cell Cycle Control if not awached properly Cell Cycle Control non disjunc on occurs major transi ons Major Transitions G1 S G1 S G2 M G2 M metaphase anaphase M to A early development Early Development no G1 or G2 No G1 or G2 No checkpoints no checkpoints Checkpoints next lecture checkpoints start start M M Spindle spindle Cyclin Dependent Kinase CyclinHDependent Kinase CDK Cyclin Dependent Kinase Cdk Cyclin Dependent Kinase Cdk 2 subunits Kinase binds cyclin must bind kinase for CDK to be kinase p34 cyclin Two Subunits Kinase p34 and cyclin Two Subunits Kinase p34 and cyclin Cyclin subunit activates the kinase Degrade cyclin to inactivate kinase Cyclin subunit activates the kinase process overview Degrade cyclin to inactivate kinase Major role in Mitosis and S phase entry 1 Kinase p34 binds cyclin Major role in Mitosis and S phase entry 2 Cyclin ac vates kinase p34 Nobel Prize 2001 Nobel Prize 2001 3 Degrade cyclin to inac vate Tim Hunt Paul Nurse Leland Hartwell kinase p34 Tim Hunt Paul Nurse Leland Hartwell Kinase binds cyclin ac vated Experimental Systems cyclin degraded b c tagged via polyubiqui na on sent to proteasome Experimental Systems Yeast Xenopus Sea Urchin major role in M phase S phase Yeast Xenopus entry Sea Urchin experimental systems yeast Xenopus sea urchin sea clam APC Anaphase Promo ng Complex turns on for anaphase to start Yeast Yeast Yeast Cell Shape Indicates Cell Cycle Posi0on CDC Mutants Yeast Cell Shape Indicates Cell Cycle Position CDC Mutants Size and shape of yeast cells changes with cell cycle a CDK experimental system CDC mutants Cell Division Cycle mutants phenotype arrests speci c point in cell cycle always stop same point in cycle mutated gene must have condi onal muta on or it will die tried to isolate temp sensi ve mutants can reveal phenotype sensi ve temp but s ll grow acceptable temp mutant gene products iden ed cloned studied led to iden ca on of many key cell cycle regulators Cell division cycle CDC mutants Paul Nurse and Leland Hartwell Phenotype is arrest at specific point in cell cycle Must have conditional mutants or it will be dead Gene products of mutants identified cloned studied Led to identification of many key cell cycle regulators TS Mutant Yeast Cell Shape Indicates Cell Cycle Posi0on Budding Yeast vs Fission Yeast Budding Yeast Saccharomyces cereviciae Budding Yeast Saccharomyces cereviciae Fission Yeast Schizosaccharomyces pombe Fission Yeast Schizosaccharomyces pombe Isola0on of CDC Mutants Isolation of cdc mutants 1 2 3 Ts mutant undergoes base change alters codon AA subs tu on makes protein less thermally stable Protein misfolds loses func on becomes unstable non permissive temp Isolation of cdc mutants Ts Mutant has base change to alter codon Amino acid substitution makes protein less thermally stable Protein misfolds loses function unstable at non permissive temperature CDK1 missing colony 35oC grow 25oC go back to 35oC study mutant Ts Mutant has base change to alter codon Amino acid substitution makes protein less thermally stable Protein misfolds loses function unstable at non permissive temperature CDK1 Gene Isola0on via Complementa0on of Mutant Transform with Library of cDNA from WT Cells mutant gene comes from rescue plasmid allows growth can iden fy mutant gene Gene Isolation by Complementation of Mutant Transform with Library of cDNA from WT Cells rescue plasmid The First CyclinHDependent Kinase CDK1 p34 similar CDC genes found in 2 yeasts CDC2 in ssion yeast CDC28 in budding yeast encodes a phosphorylated protein kinase 34 kD kinase dephosphorylated inac ve in res ng cells phosphorylated ac ve in growing cells gene c complementa on human gene