You are on page 1of 11

Carlautta Griffith

Biotech 212AC midterm

BIO212AC –Biotechnology Midterm Exam

You will be graded based upon the amount of critical thinking you put into your answers.
You need to be logical and justify your reasoning. This means your answers will not be
short ;) Cite any background research which may help you come up with or support your
ideas. Write clearly, concisely, and coherently. Organizing your thoughts is key to
thinking critically. This is not easy, some questions have no right or wrong answer (well
an incoherent irrelevant answer would be wrong), I am grading your thought process
here. You will have to look things up, this is an exercise in self exploration

You may collaborate in groups but please do your own work….copy and pasting
answers is plagiarism. Please cite all references using APA or ASM citation style.

Background- Questions 1 - 3
You are fresh out of school and just recently got a job with a new start-up biotech
company in town. You are working on a project to develop novel therapeutics to induce
apoptosis in cancer cells. You have recently observed a dramatic drop in viability of a
cancer cell line which you have been treating with an experimental compound called
Ziacid, a delocalized lipophilic cation. This cell line has several key mutations including
inactivation of p53 and a mutation creating an uncleavable Bid protein. Untreated
controls show no change in viability and the viability standard deviation between
replicates is low. Now, your boss needs you to verify some important aspects of this
experiment and produce some preliminary results which point towards a possible
mechanism for this compound in order to proceed to the next phase of trials.

When answering these questions, pretend your boss is asking you for a written
experimental proposal. Each of the following questions would be section of the
proposal.
1) You need to quickly and accurately (and cheaply) show that this change in viability is
due to induction of apoptosis and not to any other mechanisms. Explain in detail how
you would do this?

Electrophoresis would quickly and accurately (and cheaply) show that apoptosis
was effecting the cell causing it to die. It would prove this because if the cells are
undergoing apoptosis, the DNA would be all chopped up and therefore, when you run it
on a gel there will be multiple bands. This is called a “ladder pattern”. A ladder pattern
mimics a ladder that you would use to measure different particle sizes on an
electrophoresis gel. When cells undergo apoptosis, the DNA is cut into pieces and when
you run it on a gel you see a ladder pattern. DNA fragmentation by apoptosis occurs
when enzymes in the cell are released and the enzymes cut the DNA into pieces, much
like a digest. So essentially it is digested DNA that leaves that leaves the “ladder pattern”
in the gel. In cells that are not undergoing apoptosis, the DNA extracted from them form
a single band on the gel. This is how you can tell the difference. Though you should
probably check your sample multiple times because sometimes you can get genomic or
RNA blurs that might obscure multiple bands. Also, if only a small percentage of your
cells undergo apoptosis then it will be much harder to see multiple bands. Though the
threshold for visable bands is greater than 40%. So typically if you observe a band in a
particular sample you run on a gel more than 40% of that sample has undergone
apoptosis. Though sometimes if the amount is less and you have a better system for
detecting dye patterns it could be less than 40%.
citing:
"QUALITATIVE ANALYSIS OF." Home Page Del Sito Di Immunologia. Web.
08 Nov. 2010. <http://www.immunologia.unimore.it/sito ac/apobook98/chap4.htm>.

"Apoptosis/DNA Laddering Assay Protocols." Protocol Online - Your Lab's


Reference Book. Web. 08 Nov. 2010. <http://www.protocol-
online.org/prot/Cell_Biology/Apoptosis/DNA_Laddering_Assay/index.html>.
2) You accidently used media containing no serum in one of the experimental replicates
and you find that even your negative controls have undergone apoptosis; however your
treated cells initiated apoptosis twice as fast in this media than in the normal media!
(Think about what is in “normal” media) How could you explain this and how would
you test your hypothesis?

A western blot would tell you that this change in viability is due to apoptosis
because the drug attacks mitochondria which release cytosome 3 which causes a chain
reaction in caspace 3 which is a protein in the mitochondria membrane, which makes it
fall apart thereby making the cell rupture and undergo apoptosis.
Apoptosis needs RNA and energy to make proteins to start the process. It sends
out RNA which are transcribed into “pro-apoptosis proteins”, one of which is cytosome
3. Western blots detect certain proteins. I would perform a western blot on each of the
samples to detect how much cytosome 3 is in each sample and their concentrations. If the
concentrations are higher in the treated cells then I know that the drug “ziacid” is making
the cells produce the RNA that translates into cytosome 3 which causes a chain reaction
in caspace 3 which makes the cell undergo apoptosis.
Fetal bovine serum is a stabilizer for cells. It is low in antibodies so many
different types of cells can grow in it. It also has nutrients and proteins that growing cells
need. Without FBS (fetal bovine serum) many cell lines will die because there isnt
enough nutrition in the media. The reason for both the treated cells and the untreated cells
dying is because there is not enough nutrients in the media to prevent the cells from
undergoing apoptosis. Also, FBS greatly reduces the risk of over-toxicity if there is a
toxic or semi-toxic element added to the media. It basically waters down the toxicity to a
sustainable level.
Citing:
"Fetal Bovine Serum." Wikipedia, the Free Encyclopedia. Web. 08 Nov. 2010.
<http://en.wikipedia.org/wiki/Fetal_bovine_serum>.
3) You must present some data which indicates a potential apoptosis initiation
mechanism. Given your cell line genotype and your experimental drug, what would you
hypothesize? How could you test this?

There are two important mutations that are in this cell line; Inactivated P53 is one of them
and uncleavable BID is the other. Inactivated P53 initiates apoptosis when strange
divisions in the cell are taking place, ie; daughter cells not splitting, uneven
chromosomes, duplicated chromosomes. Uncleavable BID initiates apoptosis by caspase
cascade. Caspase cascade is the pathway stated in problem 2. Basically an RNA message
is sent out which translates into cytosome 3 protein which then causes the protein caspace
3 to unravel in the mitochondria's membrane making it rupture. Without it's
mitochondria's, the cell cannot produce ATP and thereby dies and undergoes apoptosis.
Because this drug initiates apoptosis, we can use a western blot to determine which
pathway is used to make the cell undergo apoptosis. If it isnt caspace cascade, then we
can test for other protein triggers by using a western blot technique. As you go through
the list of possible pathways and figure out which ones are not the trigger, you narrow
down the search to find out which is the true pathway. There are many pathways to lead a
cell into apoptosis, some of the pathways include: Extrinsic pathway, intrinsic pathway,
and caspace cascade. Although extrinsic pathway would probably be the least likely
culprit because it triggers apoptosis independently of the p53 region which is a tumor
suppressor region. When a mutation occurs in this region the cell is more likely to
undergo apoptosis. Another test would be to see if there is a mutation in the p53 region of
the cell and then derive all the pathways that come off of that.
Citing:
"Apoptosis Pathways." Genentech BioOncology Cancer Therapy Research. Web.
08 Nov. 2010. <http://www.biooncology.com/research/apoptosis/pathways/index.html>.

Bioinformatics

Background-
You are the bioinformastition for a R&D lab which works on isolating novel
proteins from environmental metagenomic samples. You have been given the following
DNA sequence fragment to analyze. Answer all associated questions to the best of your
ability. Think about your answers, do they make logical sense?

> Unknown environmental sequence


tttgaatggctgattggcagcccgcgctggcgcgaaagcgcggcggaacgcggcgatgtg
aacccggtgggcggcctggaagaagtgctgtatgaactgagcccgattgaagattttagc
ggcagcatgagcctgagctttagcgatccgcgctttgatgatgtgaaagcgccggtggat
gaatgcaaagataaagatatgacctatgcggcgccgctgtttgtgagcgcggaatttatt
aacaacaacaccggcgaaattaaaagccagaccgtgtttatgggcgattttccgatgatg
accgaaaatggcacctttattattaacggcaccgaacgcgtggtggtgagccagctggtg
cgcagcccgggcgtgtattttgatgaaaccattgataaaagcaccgataaaaccctgcat
agcgtgaaagtgattccgagccgcggcgcgtggctggaatttgatgtggataaacgcgag
accgtgggcgtgcgcattgatcgcaaacgccgccagccggtgaccgtgctgctgaaagcg
ctgggctggaccagcgaacagattgtggaacgctttggctttagcgaaattatgcgcagc
accctggaaaaagataacaccgtgggcaccgatgaagcgctgctggatatttatcgcaaa
ctgcgcccgggcgaaccgccgaccaaagaaagcgcgcagaccctgctggaaaacctgttt
tttaaagaaaaacgctatgatctggcgcgcgtgggccgctataaagtgaacaaaaaactg
ggcctgcatgtgggcgaaccgattaccagcagcaccctgaccgaagaagatgtggtggcg
accattgaatatctggtgcgcctgcatgaaggccagaccaccatgaccgtgccgggcggc
gtggaagtgccggtggaaaccgatgatattgatcattttggcaaccgccgcctgcgcacc
gtgggcgaactgattcagaaccagattcgcgtgggcatgagccgcatggaacgcgtggtg
cgcgaacgcatgaccacccaggatgtggaagcgattaccccgcagaccctgattaacatt
cgcccggtggtggcggcgattaaagaattttttggcaccagccagctgagccagtatatg
tatcagaacaacccgctgagcggcctgacccataaacgccgcctgagcgcgctgggcccg
ggcggcctgagccgcgaacgcgcgggcctggaagtgcgcgatgtgcatccgagccattat
ggccgcatgtgcccgattgaaaccccggaaggcccgagcattggcctgattggcagcctg
agcgtgtatgcgcgcgtgaacccgtttggctttattgaaaccccgtatcgcaaagtggtg
gatggcgtggtgagcgatgaaattgtgtatctgaccgcggatgaagaagatcgccatgtg
gtggcgcaggcgaacagcccgattgatgcgggtggccgctttgtggaaccgcgcgtgctg

4) Your boss wants to know the protein that is associated with the DNA fragment. Paste
the protein sequence. Is this a complete protein? Is there any secondary structure
associated with this protein, if so include a graphical representation of this.
RNA polymerase III is the protein associated with this DNA fragment. This is not a
complete protein. Graphical representation has been attached. RNA polymerase III is a
protein that transcribes DNA into ribosomal RNA so the ribosomes can translate it into a
protein. It is a very essential gene for a cell to function. If a cell has unviable RNA
polymerase III then it cannot create proteins to survive. I found out the protein by pasting
the sequence into a converter that turns it into protein code, then searched the protein
code and entered it into a site that found matches. The closest match was RNA
polymerase III. I know this is not a complete protein code because I looked up the whole
code for the protein and it is MUCH more code than what was given here. There is
secondary structure with this protein, as there is all proteins. I found a site that gives a
graphical representation, though it is not in a photograph but in a colour code that tells
you where all the twists and bends are.
Citing:
"RNA Polymerase III." Wikipedia, the Free Encyclopedia. Web. 08 Nov. 2010.
<http://en.wikipedia.org/wiki/RNA_polymerase_III>.
5) What type of protein could this be, i.e. what’s its putative function? Does it have any
conserved domains, if so what are they and what do we know about them? What could be
the protein’s function? Is there a version of this protein which has a determined 3D
structure?

RNA polymerase III is the most likely protein that this particular sequence codes for.
After doing a search and finding that there is a 99% likelihood of this being RNA
polymerase III. It's function is to transcribe DNA into ribosomal RNA which is then read
by ribosomes and translated into protein. Transcription factor BRF is a conserved domain
and it is a binding protein. A binding protein is a protein that binds to DNA. Some
binding proteins can bind to only single stranded DNA and some only to double stranded
DNA. They serve all kinds of functions like cleaving the DNA, repairing the DNA, and
acting as a port for other proteins to attach to. There should be a determined 3D structure
because if it cannot be different all the time and all proteins are 3D.

"Conserved Functional Domains of the RNA Polymerase III General


Transcription Factor BRF. — Genes & Development." Genes & Development. Web. 08
Nov. 2010. <http://genesdev.cshlp.org/content/8/23/2879.full.pdf html>.

"DNA-binding Protein." Wikipedia, the Free Encyclopedia. Web. 08 Nov. 2010.


<http://en.wikipedia.org/wiki/DNA-binding_protein>.

6) What organisms could your protein have come from? Your boss wants a preliminary
report highlighting the potential evolutionary relationship of your unknown protein and at
least 15 similar proteins. What did you find out? (Hint: think trees) Did this protein
evolve independently from a single gene? Is your given protein relatively old or young,
how can you tell?

My proteins have come from various strains of mycobacterium. Mostly


Mycobacterium Tuberculosis. It evolved from one common ancestor, although there are
clearly there are three major branches. Though they all originated from “ mycobacterium
tuberculosis”. When I looked at it in blast, there was an organism that had a difference of
50 base pairs. In a conserve domain between organisms, one nucleotide difference, we
can assume 10k years of evolutionary separation. So basically it's an old organism by this
fact.
It evolved from a single gene, not independently because there was only one
ancestor, not two. If it evolved independently it would have more than one ancestor.
Here is a picture of the tree:

You might also like