Cell Cycle and its Control Lecture 37BSCI 420,421, 620Nov 27, 2002 “ Cells come from previous cells” – R. Virchow 1.Intro to the cell cycle 2.The cell cycle control system a. MPF in frogs b. Cdc mutants in yeast c. The mammalian cell cycle

1.Intro to the cell cycle When you look at cells under a microscope, you see 2 states: Dividing cells and cells in an Interphase between divisions. With the introduction of DNA labeling methods like 3H-TdR and ARG or BrdU and Fl-anti-BrdU-DNA, 4 phases were IDd.

These two methods allow progression thru the cell cycle To be studied. Eg The Labeling index after a pulse tells you how many cells are in S (a fx of how fast/many cells are cycling). %S = tS/tT if all cells cycling %M= tM/tT “ “ “ (Fig )

Flow cytometry gives a histogram of no. of cells vs. Amount of DNA/cell. Gives % cells in cycle phases.

The cell cycle is a set of biochemical events driven by a control system that tells cells whether they can enter the next phase of the cell cycle.

How does this CC control system work? By activation and inactivation of a set of protein kinases called Cyclin-dependent protein Kinases (CdKs). The two key components are Cyclin, a protein whose levels go up & down during the cell cycle, and the CdK enzyme itself.

1.The cell cycle control system a. Maturation Promoting Factor (MPF) in frog eggs Exposure to male Gonadotropin (Pit) Progesterone (Foll. Cells)

Masui & Markert found that a small amt. of cytoplasm from mature, metaphase-arrested eggs injected into interphase- arrested immature oocytes induced meiotic divisions and Maturation to metaphase II. (in absence of progesterone) This factor they named MPF MPF Act imm Meiosis Meiosis Mit 1 Mit 2 oocyteI IIfert time

Using this activity when inj into imm oocytes as a Bioassay, M & M purified MPF down to 2 proteins: A 34 kD protein kinase that stays relatively constant & A 56 kD protein that increases and decreases during cycle. They named the cycling protein “Cyclin” & the enzyme CdK.

b. Cdc mutants in yeast Yeasts are single-celled fungi with cell cycles very sim to ours. 2 diff species have been extensively studied:

Temperature-sensitive mutants can be selected that arrest in Particular phases of the cell cycle when switched to restrictive (high) temps. These are called cell-division-cycle (cdc) genes.

E.g., when grown at restrictive temp the normal yeast (A) Show all cell cycle stages The cdc 15 mutants (B) Are blocked at the end of M

Yeast mutations demonstrated genes for: One Cdk enzyme Three classes of cyclins: G1/S-cyclin, necessary for cells to pass the Start point & enter S S-cyclin, required to initiate DNA replication M-cyclin, required to enter Mitosis Several proteins that regulate Cdk activity: CAK, a Cdk-activating kinase

Also, Wee 1 a kinase that inhibits Cdk by P04ing a tyr, & Cdc 25, a phosphatase that stimulates Cdk by removing that P04 p27 a direct-binding inhibitor of Cdk

c. The mammalian cell cycle Human cells were fused together in diff CC phases:

Overview of cell cycle checkpoints: