Download Cell Cycle and its Control

Cell Cycle and its Control
Lecture 37
BSCI 420,421, 620
Nov 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. 17-11)
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 interphasearrested immature oocytes induced meiotic divisions and
Maturation to metaphase II. (in absence of progesterone)
This factor they named MPF
MPF
Act
imm
oocyte
Meiosis Meiosis
I
II
fert
time
Mit 1
Mit 2
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: