Download doc Cell Cycle 1 Notes (Pause)

Cell cycle in a unicellular organism is:
1. Chromosome replication and cell growth
2. Chromosome segregation
3. Cell division
a. The cell won’t divide if not in the right environment! (ex: too cold)
Cell cycle is conserved across the species, so one can study yeast to know about humans.
Yeast is also chosen because it divides very quickly (once ever 20 minutes!) However,
this is also place for a major difference between humans and yeasts: yeasts don’t stop
dividing once it is in the right environment (food and temperature), while our cells do not
divide 99% of the time despite the fact that they are always in the right food and
temperature. The speed of yeast development should be compared instead to the
development of embryonic development. But once development is over, the cell stops
dividing and stay around in G1/G0, performing its functions without division. Some
exceptions are when there are huge death tolls around it (i.e., blood cells or a hang-over);
in that case, cells have to replicate quickly in order to re-establish equilibrium. This is
mainly because the cell is 100% focused on dividing when dividing, so it can’t do its job
when it is dividing.
A few decades ago, to look at the cell, the only visible “eras” were mitosis and
cytokinesis, if one stained the DNA and watched for the pulling apart of it during
anaphase.
DNA-replication = S-phase = longest phase (12-18 hours) because the cell has to
replicate the DNA (which is quite long).
M-phase = segregation = shorter phase (1-2 hours) Spindles form during prophase,
microtubules attach to the centromeres of the chromosomes during metaphase. Each
chromosome has to be attached to the microtubules for the cell to divide; the cell does not
divide if the chromosomes are not attached.
M-phase = mitosis (nuclear division) + cytokinesis (cytoplasmic division)
Gap-phases = G1 and G2 = between M-phase and S-phase; there are variable between
cells and organisms. For example, embryos have almost no gap phases since they have to
divide rapidly (especially if outside the mother—ie, frogs, which are prone to be eaten).
Gap phases are basically phases to take a break and check if everything is okay. G1 is to
check for extracellular environment (after M-phase), because S-phase needs a lot of
energy and time, so it is important to have a good environment. In G1, the cell is “ready
to go” into S-phase. A cell in G1 for a long time goes into G0 phase, in which the cell
completely disassembles its cell cycle machinery (not expecting another S-phase). A cell
in the cell cycle is full of cell cycle proteins (so only focused on replicating DNA and
dividing); disassembly of these proteins allow for normal function and making of
functional proteins. If there is a need for replication, the cell goes back into G1 phase to
build up its cell cycle proteins.
G1 is important for oncology. If there is a mutation in the cell and something goes wrong
with G1, the cell can’t sense if it should continue dividing so the extracellular
environment has no effect on it.
G2 is important for sensing the stability of the DNA (G1 too). If there is something
wrong, it does not go into the next
phase (M for G2, or S for G1) until the
problem is fixed, e.g. the cell is back to
wild type. G2 and G1 might last up to
hours if there is anything wrong with
the cell. Organisms like yeast do not
care much about G1 or G2, as there are
new daughter cells every 20 minutes
and one wrong cell won’t cell a colony.
We like to use yeast in studies because
(1) its genome is small and fully
sequenced, (2) its generation time is small (20 minutes) (3) has a similar cell cycle to
humans, (4) it is very easily manipulated—so simply worked with, and (5) it is haploid,
so it is easy to knock out genes (knocking one is enough—no need for two). Additionally,
all yeast-human proteins are conserved.
On the other hand, yeast is not entirely the same as multicellular organisms, which have
much more complicated cell cycles—different tissues have to divide in different times,
etc.
To study yeast in the 1970s, there were two tricks:
1. Randomly make mutants (via UV, carcinogens, etc.)
2. Mutated proteins become temperature sensitive (i.e. defective at higher
temperatures)
a. Therefore, the phase at which the proteins work will not continue; for
example, if the mutated protein was at the S-phase and temperatures were
risen to 42oC, all mutant cells will stop at G1, or cytokinesis (in which
case, when looked under the microscope, all cells will seem to be in the
midst of pinching off)
b. Pick the mutant yeast out of the culture and regrow at functional, lower
temperatures to have a colony of mutants
3. Sequence DNA and find DNA of the defect
a. This might be complicated, because there might be mutations all over the
place (silent mutations, irrelevant mutations, etc.)
b. Smarter method would be to re-introduce the wild-type genome by
transfection (clone w+ yeast genome into a library of plasmids and
reintroduce the plasmids to the bad yeast) and check which one is rescued
and will go back through the mutated phase
c. Separate the one that is rescued and separate its plasmid DNA; sequence
the plasmid DNA
All cell-division cycle genes (CDC genes that regulate cell cycle) were found in such a
way. Mammalian CDC genes were also found and were conserved to yeast genes.
Oocyte zynopus eggs are 0.5mm and used as model genes. They are large and isolatable
(used to growing outside), and consequently favorable for in vitro studies. The reason
they are large is evolutionary—they do not want to be digested by predators, so the eggs
grow (without division) to 100,000X in months (so the cytoplasm is 100,000X big, but
the nucleus is still that of the original cell). Then, after fertilization by the sperm, the
humongous cell divides incredibly quickly (~5,000 divisions in 7 hours—15 minutes for
each division; no G1/G2 phases) in order to make a tadpole (to escape predators, once
again). This is highly specialized and does not happen in mammalian cells since
mammalian embryos are inside.
Experiments include putting the cytoplasm from activated frog eggs, ATP, nd nuclei from
frog sperm in a test tube and subjecting to warm temperatures. The entire cell cycle can
be seen in the test tube. Running the extract from the test tube in columns can separate
out the proteins inside. Oocytes are more “biochemical” in this aspect than yeasts (which
are more “genetic” in that you have to work with genes and knocking out and mutating).
Another reason to study multicellular models is that they are prone to cancer—i.e. have
highly regulated systems saying “don’t grow” and “don’t divide” to many different types
of tissues, whereas yeasts don’t get cancer. Hence, multicellular organisms get tumors
when the control fails.
Mouse embryos can also be studied because they are available in large quantities and can
have cells isolated. One technique is to extract mouse embryo (at high availability), mash
it up, and culture it on a Petri dish. Often, fibroblasts end up growing. The M-phase is
always round and ones in G1/S/G2 are flat and round.
Before, people counted mitotic index (counted the number of “round” cells in mitosis and
got a percentage out of the total number of cells). Now, people have new methods to
study cell culture (cells in petri dish) by taking out nutrients/temperature/etc. and
observing when cells arrest (in G1/G2/etc). This might be studied by radioactive assays
(incorporate radioactive nucleotides into a cell—which would only be used during S
phase). Then, count cells again and how many cells in S phase could be known. This is
how you tell if a cell is proliferating.
Another type of assay is through modified nucleotides (i.e. bromo-deoxyuridine, a
modified UTP), which is also incorporated into the DNA. Now add a monoclonal antibody linked to a fluorescence molecule.
More recently, people have began using flow psytometry, or FACS (fluorescence
activated cell sorting). Psytometry means cell measurement, so the number of cells and
amount of DNA per cel can be measured
and plotted. You take a cell, add a
fluorescent dye, shine a laser on it, and get
a detector that measures the intensity of
the shine (proportional the amount of
DNA in the cell).
Therefore, the amount of DNA in cells is
from 12 (2 for G2, after S phase, and 2
in G1, after M phase). Now experiments
can be done and it can be seen which peak
goes higher (so the arrest phase can be
seen). Of course, there might be more or
less than 12 DNA; this is because it is
an average taken. Additionally, when cells
die, they digest/cleave their own DNA—
so there might be a large peak at <1 (for
apoptotic cells).
For example, if DNA is damaged during
S-phase, G2 numbers will probably rise,
while if nutrients were lowered, G1
numbers will probably rise.