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Lecture 22
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Localizing the HPRT and the TK gene
It’s possible to fuse the cells of 2 organisms and generate a hybrid. We also have to kill the parental cells,
because those will be the ones that are present as a majority in petri dishes.
o We need to use markers to kill these cells.
o We need 2 markers, one for each parent organisms.
o Both organisms need to a have missing phenotype to eliminate the parental cells.
o HPRT and TK gene: markers that are part of the enzymatic machinery to synthesize DNA.
o We need to put the drugs to stop synthesize the DNA in major pathway.
o Cells can synthesize DNA in minor pathway with TK genes and HPRT genes.
How to locate the genes on chromosomes?
o We need a rat cell without those genes. (note that the genes must code for proteins that can
complement each other, despite coming from different organisms.
o Where is the TK genes? You need a mouse cells with TK-, you need to complement the human
cells with the TK+ gene.
o HPRT- results in death.
o Only hybrid cells will survive in HAT mediums.
o Only hybrid cells will grow that have the human TK+ gene.
o And by looking at these progenies we see that they contain chromosome 17, so in this case we
known that the TK+ gene on chromosome 17.
o When you make a hybrid with a human and rat cells, the rodents win, after division, such as cell
is quite big, will have 80 chromosomes, when those cells are dividing, we will lose most of
human chromosomes.
 So we only end up with one or 2 chromosomes.
 All the cells that have chromosome 17 will survive.
o We do the same thing with HPRT-, all the hybrids contain human X survive.
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Without sequencing we have identified 2 genes on the human chromosomes.
Rescuing a defect: differentiatin human and roden gene products
o We used this to locate an enzyme with UMP kinase: we again need a mouse that have deficient
gene for that enzyme. Actually we don’t, because the enzymes are different enough to be
detected on gels.
o Different electrophoretic mobility of gene products combined with detection system: UMP
kinase.
o Mouse and human UMP kinase differ by few amino acids, which affect the migration of the
enzyme on gel.
 Enzyme activity assay or Western blot.
o Mouse cells were UMP-kinase negative.
o Where is human UMP-kinase?
 The chromosome has to be present everytime we see the activity, and absent if there is
not activity.
 We probably will have an exam on this.
 By analyzing 8 kinases, we find gene on chromosome human 1.
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Mapping a measles virus receptor gene
o Human can be infected by measle virus, but not rodent cells.
o Human cells have the receptor.
o T(1,21) translocation between 1 and 21
o However this doesn’t mean that 1 is intact.
o Indeed human 1 does contain the gene of interest, the 4th hybrid tells us that the the 4th bybrid’s
translocation defected the gene of interest.
 So in this case we can even tell where the gene is on the chromosome, by looking at the
chromosome.
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Microcell hybrid fusion.
o In which chromosomes are transferred individually is still used in Montreal to identify defective
genes implicated in neurological disease.
Lecture 22-23
Cancer genetics and gene therapy
During mitosis, those cells are not normal, they don’t divide according to normal rules. You will have
breaks in those chromosomes. You are likely to generate fusion between the chromosomes.
Incidence of cancer:
o 1/2.2 in males, 1/2.5 in females.
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Pancreas cancer is most deadly
Cancer between different populations are occurs different.
o Genetics link and environmental link.
o Environment is a big influence, (because immigrants within 2 generations, the cancer type they
have also changes, due to lifestyple changing)
An overview:
o Note the cells gets different signals including:
o survival cues, proliferation cues,
o death cues and growth-inhibition cues.
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Note that in cancer cells have deathcues off.
o They can generate their own proliferation cues, survival cues
o What we will get is uncontrolled survival and proliferation, we will get tumor.
Cell cycle:
o For a cell to go to cell cycle, the mitosis will only take 1 hour, most of the time, the cell is
preparing for mitosis.
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To P or not to P” : That is the kinase question
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o Active-> inactive
o Inactive -> active
Cyclin binding to CDK activates CDK which phosphorylate target proteins that function to either inhibit
or activate its function.
What about death?
o Normal cells can commit suicide, called apoptosis or programmed cell death. Specific enzymes
are involved in this pathway. We will be able to see the fragments.
o Nice thing about apoptosis, is that there is no fragments released in cytoplasm, so inflammation
no attraction of antibodies.
o If apoptoic pathway is active, the cell will die.
o Cancer cells must inactivate the pathway,
How does apoptosis work?
o Cells need to make an enzyme called caspases, which is an executioner.
o That caspase is inactive in the intact form, you need to fuse the 2 portions together, you need to
cleave the capsase at 2 points, which give a large and small subunit that interact with each other
to fuse into an active form of the caspases.
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o The active caspases proteloyze (digest0 target proteins.
 Which inactivate the DNA endonuclease-sequestering protein that results in DNA
fragmentation.
 This leads to activation of actin-cleaving protein that results in the loss of normal cell
shape.
 Breakdown of organeels and fragmentation of cell.
Properties of cancer cells:
o Give them the food, let them grow. Cells will grow, divide divide indefinitely.
o They are immortal, we work with cancer cells that have been isolated long time ago.
o Normal cells: In the beginning they will grow, they enter a crysis, (Grow and sinesis.)
o Anchorage dependence: cancers, they don’t need a media to grow. Normal cells need a surface
to grow and cannot grow on top of themselves.
o Metastatic potential: the spread of the disease from one organ to another. In addition cancer
cells are not contact inhibited, it will break through membranes to invade other cells.
 Cancer cells can break through the membranes.
o Angiogensis: formation of blood vessels to cancer cells, the food is delivered to them.
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o Needs a lot of irradiation to kill a cancer cell than normal cells.
Car analogy:
o Gas pedal=positive regulators= oncogenes
o Breaks=negative regulators=tumor suppressor genes
o Mechanics= repair mechanisms= DNA mismatch repair mechanisms.
Oncogenes
o Genes encoding positive regulators of cell growth.
o The first oncogene (v-src) in retrovirus.
o V-onc can cause cellular transformation
o V-onc are related to normal cellular genes (proto-oncogenes) which are involved in growth
control or signal transduction mechanisms.
In each of us, we have the equivalent of those genes.
Viral oncogenes vs cellular counterpart (c-onc)
o Same gene present in our DNA, cellular DNA have introns, v-onc don’t.
o C-onc encode proteins that can be in a active or inactive state, v-onc are always in active form.
o V-onc escapes the cellular control mechanisms that the c-onc is subjected to.
Non-viral oncogenic transformation
o In humans, viruses rarely give cancers. Not major way.
o The cellular onco gene in absence of viral contributions is activated or inactivated by mutations
or inappropriate expression (loss of regulation, chromosome translocation).
o Retroviral oncogenes: an “inhuman thing”?
o Well-characterized oncogenes and functions of the corresponding proteins.
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How to find those oncogenes, they isolate the DNA from cancer cells. They added marker(red) to each
fragment.
Transfer DNA into normal cells. Oncogene integrates into cell and transforms its descendants into
cancer cells.
A gene c-H-ras oncogene is implicated in human bladder cancer.
They isolate specific DNA gained by transformed cells (identifiable because it has the marker).
They transform normal cells with the isolated specific DNA and find that the result is cancer in those
cells.
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