Exam 3

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

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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

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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

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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

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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

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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

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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

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Section 4 (continued if necessary)

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Evening Core 2008 Exam 3; 02/20/08

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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

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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

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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

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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

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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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