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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Notes to Students:
1.
Please place all coats, books, papers, cell phones, pagers, other electronic devices
at the front of the room. Please sit at least one row and two seats away from your
classmates. Now is the time to use this cavernous space.
2.
Write your name on EACH PAGE. The pages will be separated for grading. If
your name is not on the page and we cannot identify your work you will not
receive points for that page. You don’t have to write your name on this page.
3.
There are 6 sections. At the end of the exam, please separate the sections and put
them in 6 piles in the envelopes in front.
4.
Space for each question is provided on the FRONT of the exam pages. You
should not need to use the extra space on the back of the page. However, if you
make a mistake on the front, you may cross it out and write your answer on the
back.
5.
Answer completely, but CONCISELY and clearly. One or a few words may be
enough for some questions. Extraneous, unnecessary information will not gain
you points. If the extra information is wrong, then it may cause points to be taken
away. Diagrams, flow charts, and pictures are acceptable in addition to or in lieu
of text.
6.
The points on the exam add up to more than 100. The scores will be adjusted and
expressed as percent correct.
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 1 (20 points):
1.
Name a type of RNA present in eukaryotic cells. Give one of its functions in the
cell. Also state which RNA polymerase is responsible for its synthesis.
2.
On what type of RNA is an RNA cap found? Name one of its functions.
3.
Name one difference between a weak and strong promoter.
4.
Name two differences between RNA and DNA.
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 2 (20 points):
RNA processing questions (each question worth a total of 2 points; questions #4-5, each
part worth one point)
1. Which answer best describes RNA processing?
a. the process by which RNA is assembled from a DNA template
b. the attraction of a binding protein and other transcription factors to tell the
RNA polymerase where to bind and begin making RNA
c. the series of changes that must take place on an RNA molecule before it can be
called “mature”
d. the stepwise addition of amino acids to a growing polypeptide chain
e. the folding of a polypeptide chain into a specific three-dimensional structure
2. A gene for collagen possesses 40 exons. How many introns does it possess?
a. 38
b. 39
c. 40
d. 41
e. none of the above
3. The excision of introns from pre-mRNA in eukaryotes is carried out by:
a. Ribosomes
b. Ribozymes
c Lysozyme
d. Spliceosomes
e. Ribonuclease A
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
4a. Name one tRNA modification that must be performed on pre-tRNA.
4b. Name one rRNA modification that must be performed on pre-rRNA.
5a. What does the abbreviation “UTR” stand for?
5b. Name one defined RNA element that could be found in a mammalian UTR.
6. Why is unprocessed pre-mRNA not normally found in the cytoplasm?
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
For the following questions (7 through 10), please refer to the following diagram of a
mammalian pre-mRNA, and to the data on the Northern blot shown below.
AAUAAA
Ovary
Testis
Pancreas
UAA
AAUAAA
UAA
Spleen
Cardiac muscle
Smooth muscle
Neural tissue
Markers (kb)
AUG
CAA
Poly A+ RNA was selected from each tissue shown, was separated on an RNA gel which
was transferred to a nylon membrane for a Northern blot. The probe was complementary
to sequences in exon 1. Each exon ~ 1kb, each intron ~ 1 kb. This gene is processed
differently in different tissues.
5
4
3
2
1
Choose one answer for each question.
7. No mRNA is found on the Northern blot from the pancreas for this product. Assuming
that the gene is transcribed, what is the best possible reason or reasons for this?
a. the mRNA was subjected to NMD
b. C-to-U editing occurred
c. The mRNA was not made
d. Answers (a) and (b)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
e. None of the above
8. The mRNA product made in the cardiac muscle and testis is most likely the result of
a. Alternative splicing
b. Alternative polyadenylation
c. Normal i.e. NOT ALTERNATIVE processing
d. C-to-U editing
e. None of the above
9. The mRNA product made in the smooth muscle, spleen, and ovary is most likely the
result of:
a. Alternative splicing
b. Alternative polyadenylation
c. Normal i.e. NOT ALTERNATIVE processing
d. C-to-U editing
e. None of the above
10. The mRNA product made in neural tissue is most likely the result of
a. Alternative splicing
b. Alternative polyadenylation
c.
Normal i.e. NOT ALTERNATIVE processing
d.
Answers (a) and (b)
e.
None of the above
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 3 (20 points):
(Answer any 2 questions: 2½ points for each part, total 5 points).
A.
Which two enzymes are likely to produce the most fragments for any given
DNA molecule?
B.
Which enzyme(s) is (are) not likely to cleave a 50 kb molecule even once?
C.
Which two enzymes produce sticky ends that bind to each other?
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
2.
Transgenic Yfo’s are the easiest transgenic animals to make since plasmids fed to
them are taken up by every cell and integrated via a retroviral mechanism into the
genome where they are maintained stably. The Yfo shown below contains a mutation in
the Straight knee (STK1) gene causing its knees to stay permanently bent as shown. This
gene is normally expressed in bone cells. Your colleague has mailed you a PCR
fragment with the coding sequences from the WT Straight knee gene and has asked you
to genetically cure a mutant Yfo back to WT.
DNA parts for questions A & B below:
Elements may be used more than once per plasmid!
E. coli ORI C
Yeast ARS (origin of DNA replication)
Yfo origin of DNA replication
Yfo nonspecific enhancer of transcription
Bent kneed mutant (stk1-) Yfo
Yfo bone-specific enhancer of transcription
Yourfavoritii organismii
STK1 ORF (missing UAA at end)
STK1 ORF (WT sequence)
STK1 upstream controlling region (DNA upstream of YFG ATG)
STK1 downstream controlling region (DNA downstream of YFG UAA)
Green Fluorescent Protein coding region
Beta-galactosidase coding region
Beta-globin introns
Actin protein encoding DNA
Multiple cloning site (polylinker or MCS)
DNA encoding 6 Histidine residues
Flu virus haemagluttinin protein DNA
DNA encoding a bone specific repressor of transcription.
E. coli DNA transposon
E. coli selectable marker gene (Amp or Tet resistance gene)
Yfo selectable marker gene
Yeast selectable marker gene (URA3 or HIS3)
Yfo tumor virus coat protein
Yfo tumor virus LTR (long terminal repeat)
Yfo papillomavirus
Bacteriophage lambda repressor
Yfo acetlycholinesterase gene
Yfo rhodopsin gene
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
A. Using the DNA parts listed above, design a recombinant DNA plasmid that when fed
to mutant Yfo would likely cure this condition. Display your answer on a circular
plasmid showing exactly where each element is located making sure sequences are
properly oriented with respect to gene polarity. (10 points)
B. Design a second plasmid that could be used to show that the STK1 construct you have
introduced produces a fluorescent protein that is correctly expressed (5 points).
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 4 (20 points):
Explain the significance of FIVE of the following for mammalian protein synthesis.
(Each answer is worth up to 4 points and should be no more than several sentences
long, with a diagram if appropriate.)
1.
2.
3.
4.
5.
6.
aminoacyl-tRNA synthetase
tRNA
monocistronic mRNA
peptidyl transferase
release factor
signal recognition particle
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 4 (continued if necessary)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 5 (10 points):
1. The diagram below shows the interconversion of the 20S proteasome core particle and
the 26S proteasome core particle-plus-regulatory cap, both drawn in cross-section.
a. State one advantage of the core particle barrel-like structure for the cell. (2 pts)
b. State two functions of the regulatory cap. (3 pts)
Nature Structural Biology
7: 1062 2000
2. The figure at the right shows a diagram of the ubiquitin-proteasome pathway.
a. The first reaction uses ATP to activate ubiquitin. Identify another step in the pathway
that uses ATP. (2 pt)
b. If you wanted to target this pathway to treat a disease, which step
would you choose to inhibit to achieve greatest specificity and why?
(3 pts)
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BiochemSocTrans 32: 724 ‘04
Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 6 (20 points):
1. You join a Drosophila melanogaster lab and begin using a new drug FMK that
causes a severe defect in Long Term Memory. The signaling pathway that is
responsible for Long Term Memory in D. melanogaster is conserved in the yeast
Saccharomyces cerevisiae, although in yeast its inhibition results in growth arrest.
As predicted, yeast cells stop growing when FMK is added to their growth
medium.
i) If you wanted to take a genetic approach to identify the biochemical target
of FMK (and your mentor could convince you that the targets would be
identical in yeast as in D. melanogaster), why would you use yeast rather
than D. melanogaster? Remember to provide your reasoning. (4 pts)
ii) Outline the steps you would take to identify yeast mutants that no longer
respond to (i.e. they now grow in the presence of) FMK. Remember to
include the steps that would allow you to determine the number of genes
affected. (8 pts)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
iii) At least one of the FMK resistant mutants has a recessive mutation.
Outline the steps that you would take to isolate the wild-type copy of the
gene that is altered in that mutant. (6 pts)
2. Why can evolution proceed faster with meiosis than with mitosis? (2 pts)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 7 (10 points):
In fruit flies, red eye color is dominant over white eyes and long legs is
dominant over short legs. A DIHYBRID red eyed, long legged fly is mated
with a white eyed, short legged fly. Among the 100 offspring, you find 40
with red eyes and long legs, 40 with white eyes and short legs, 10 with red
eyes and short legs and 10 with white eyes and long legs.
5 pts. Are the eye color and leg length genes genetically linked? Explain
5 pts. What is the genetic distance between the two genes and how did you
arrive at this result?
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
Section 8 (20 points):
1. You have isolated liver cells from a mouse and cultured them in the lab. To study the
function of a particular liver enzyme, you insert the gene encoding the liver enzyme into
an expression vector and insert the vector into the cells in culture. You then surgically
transplant the modified cells into a mouse liver to study liver function with the presence
of the modified cells. Is this mouse that has received the transplanted liver cells
considered a transgenic mouse? Why or why not? (2 pts)
2. What type of promoter would you use for a transgene construct for pronuclear
injections (random insertion) if you wanted expression of your gene in all cells of the
body? (2 pts)
b) What if you wanted expression only in the beta cells of the pancreas? (2 pts)
3. What are two main problems with random insertion of transgenes? (2 pts)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
4. Briefly explain how a dominant-negative transgenic approach can create a “loss of
gene function” without disrupting the endogenous gene. (2 pts)
5. The growth factors IGF-I and IGF-II bind with high affinity to the IGF type I receptor
(IGF-IR). The actions of IGF-I and IGF-II on fetal growth were determined through
analysis of mice carrying single and combinatorial deletions of the genes for the two
ligands and the receptor. The following table shows the birthweight phenotypes
associated with these mutant lines.
Deleted Gene(s)
Birthweight (% of Normal)
Neonatal Viability
Igf-I
60%
Mostly lethal
Igf-II
60%
Viable
Igf-Ir
45%
Lethal
Igf-I/Igf-II
30%
Lethal
Igf-I/Igf-Ir
45%
Lethal
Igf-II/Igf-Ir
30%
Lethal
Data in the table provide genetic evidence that IGF-II acts on fetal growth by binding to a
second signaling receptor. Experiments from other laboratories have indicated that IGF-II
can bind to a variant form of the insulin receptor (InR-A).
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
a) Design experiments using transgenic or knock-out technology to determine the
function of the InR-A. Be specific about the strategy, the vector design and the type
of insertion you will use.
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
b) Describe how you would perform a genetic test similar to those designed in the table
above to determine whether IGF-II but not IGF-I promotes fetal growth through the InRA. Include predictions of the expected phenotypes. (5 pts)
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Evening Core 2008
Exam 3; 02/20/08
Name (please print clearly)_________________________
c) How would you test whether IGF-I functionally compensates for IGF-II in the IGF-II
null mutant? (Discuss strategy and predicted results only.) (4 pts)
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