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p53 mutation help?

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p53 mutation help?

Postby goodyearkl » Sun Apr 11, 2010 3:20 pm

Any ideas with this one? It's a part of an optional assignment I have been working on for extra credit. Everything else is complete and I have been milling this one over for about a week without coming up with a solution that I am confident in.

I will post the full question for context sake but there is really only one part of it that I am having trouble with.

You are studying a heritable form of cancer that is due to a mutation in the p53 gene. The
mutation is thought to be a deletion of 200 bases in the middle of the gene.

i) Design an experiment that would allow you to identify the wildtype and mutant alleles
from a small sample of DNA.


Can I just use a standard microarry experiment to look at global gene expression in mutant vs. wild?

ii) You are given a number of tissue samples from an affected male individual which you
analyse with the test you designed. How many wild type and how many mutant p53
genes would you expect to find per cell in non-cancerous somatic cells, tumour cells and
sperm cells?


This is the part I can't really venture a guess at. Does it have something to do with the idea that there are two alleles so there are two chances for the gene to be knocked out? How do I count them?

iii) You determine that loss of heterozygosity has occurred in the patient’s tumour cells.
List all possible cellular events that could have triggered this process to occur.


Does this mean all events that could have occured for p53 to be deactivated.

Thanks so much in advance. I normally have no problem with this stuff but this question really threw me for a loop!
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Postby JackBean » Sun Apr 11, 2010 4:42 pm

i) standard DNA chips probably won' t discriminate between your two variants. You could use qPCR or Southern

ii) having at least one functional copy of p53 drastically reduces the chance to get cancer, so in healthy cells one would expect at least one healthy allele, whereas in cancerous cells you will probably have two wrong alleles
(although cancer can have more causes, so you can have even two healthy copies of p53, but if everything else messes up, you will get cancer;)

iii) that sounds, like if you had homozygous now (that is, you don't have two, although ill alleles), so there had to be homologous recombination.

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Re: p53 mutation help?

Postby goodyearkl » Sun Apr 11, 2010 4:46 pm

Wow. That's awesome thanks. :D qPCR makes sense, but what could I use as a normalizer?
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Postby JackBean » Sun Apr 11, 2010 5:39 pm

Anything, what is usually used, actin, rRNA, GAPDH etc.
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Postby goodyearkl » Sun Apr 11, 2010 9:33 pm

Yeah thanks. That makes sense.

Would this be correct?

Non cancerous somatic cells: 2 or 1 copies of wild type p53. 0 or 1 copies of mutant p53.

Tumour cells: 0 copies of wild type, 2 copies of mutant p53.

Sperm Cells: Not sure. It says from an affected male but is p53 linked to one of the sex chromosomes? Since the male is affected would the sperm have 2 mutant copies since the mutation is heritable ? Or would the sperm be affected?
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Postby JackBean » Sun Apr 11, 2010 9:42 pm

http://www.ncbi.nlm.nih.gov/gene/7157
it's on chromosome 17.
If the man got the cancer, he probably had to have already one wrong copy and the other mutated randomly*) (it's very low chance, that both copies will mutate in one cell), so unless the man has cancer of testis or something like that, the sperms should have 1 : 1 chance to get healthy and ill allele

*) thus in the healthy cells you should get 1 healthy and one ill copy ;)
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Postby goodyearkl » Sun Apr 11, 2010 9:45 pm

Thank you so much! :D :D :D
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Postby MrMistery » Sun Apr 11, 2010 10:33 pm

i) you don't need qPCR, just regular PCR with primers that would amplify the middle of the gene. Then sequence the PCR product and see if you have the truncated product or not.

ii) Because you are studying a heritable cancer, it means that ALL the cells in the body inherit one bad mutation (the truncated gene). That's how heritable cancers work: usually it takes two mutations in the same cell line to inactivate both copies of the tumor suppressor (in this case p53). In a heritable cancer you already have one disfunctional allele, so all you need is one mutation. Think of it like this: chance of one mutation is 1 in a million. Chance of getting two is 1 in a trillion. That's a very very small number, which is why most people don't get spontaneous cancer. Now one in a million is not that small of a chance, considering how many cell divisions are in the human body.
Bottom line: every healthy cell still has one bad and one good allele, tumor cell has 2 bad ones.

iii) Homologous recombination is one possibility. Others are any other mutations that would inactivate the p53 gene: deletions, insertions etc
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Re: p53 mutation help?

Postby goodyearkl » Mon Apr 12, 2010 2:45 am

That makes a lot of sense. Thank you. :D :D

How does one explain why in tumor cells all of the genes on the chromosome towards the telomer of your gene seem to have also been reduced to homozygousity but that is not true of the genes closer to
the centromere?

It's not one of the questions I am submitting for the assignment (we only had to do so many) but I am curious about it.
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Re: p53 mutation help?

Postby JackBean » Mon Apr 12, 2010 9:47 am

MrMistery wrote:iii) Homologous recombination is one possibility. Others are any other mutations that would inactivate the p53 gene: deletions, insertions etc


But in setting he has:

goodyearkl wrote:iii) You determine that loss of heterozygosity has occurred in the patient’s tumour cells.


IMHO it's highly improbable, that the same mutation occurred by itself, without recombination.
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Postby MrMistery » Mon Apr 12, 2010 11:51 pm

i suppose technically you're right - if you wanna be specific loss of heterozygosity means that you would end up with two identical bad alleles, and the only way that would happen would be recombination.
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