BSCI 420, 421, BIOL 620 CELL BIOLOGY Fall 2002

Exam I

Name: ____________KEY__________________

Course Section: (Please circle)

420, 421 Tues lab, Wed lab, Thurs Lab, 620

Check to see that you have 8 pages, including this one.

You have 55 min. to complete the exam.

Please write clearly and in pen if you want a regrade.

Good luck.

Multiple Choice

1. ______ of 27 points

Short Answer

2. ______ of 33 points

Experimental design

3. _____ of 20 points

Essay. Write on one of these two (20 points)

4. ______ of 20 points

5. ______ of 20 points

Total _100__ of 100 points

BSCI. 421, 420, 708A Name ____________________________

Exam I, Fall 1999

(27) 1. Multiple choice: (circle the number of the one best choice-3 pts each):

a. The first living cells on earth are now thought to have had genomes that were

1) protein

2) DNA

3) RNA

b. Organelles like mitochondria and chloroplasts are thought to have originated in eukaryotic cells

by a process called

1) Phagocytosis

2) Endosymbiosis

3) Endocytosis

4) Autogenous formation

c. Chloroplasts are thought to have originated from symbiotic

1) Archaebacteria

2) Green algae

3) Aerobic eubacteria

4) Cyanobacteria

d. Mitochondria and chloroplasts resemble bacteria in that they

1) Have their own DNA

2) Have their own ribosomes

3) Reproduce by simple division in two

4) All of the above are true

e. The first enzymatic step in making a cDNA uses the enzyme

1) EcoR1 nuclease

2) Taq polymerase

3) DNA polymerase

4) RNA-dependent DNA polymerase

f. Helix-turn-helix proteins are mirror-image dimers with DNA-recognittion alpha helices spaced because:

1) Two alpha helices side by side will have their centers 34 A apart.

2) The major grooves of DNA are 34 A apart.

3) The distance from the major groove to the minor groove of DNA is 34 A.

4) Mother nature likes prime numbers

g. A poly-A tail is added to a mRNA by

1) RNA polymerase, which reads a string of complementary dTs at the end of the gene.

2) Poly-A polymerase, which adds A’s sequentially to the end of the transcript.

3) Poly-A transferase, which adds a pre-made string of A’s to the end of the transcript.

4) Poly-A correctase, which recognizes an AAUAAA signal at the end of the message and changes the U to an A.

h. Steroid hormone receptors recognize and bind to specific DNA sequences by

1) Zinc fingers

2) Helix-loop-helix domains

3) Helix-turn-helix domains

4) Leucine zipper domains

i. Release of DNA-bound RNA polymerase II to initiate transcription appears to be the direct result of:

1) Unwinding of DNA by helicases.

2) Binding of general transcription factors to the polymerase.

3) Phosphorylation of RNA polymerase by a protein kinase.

4) Removal of nucleosome(s) occupying the promotor site.

(33) 2. State the functions of:

(give the number of different functions requested for 3 points each)

a. Telomeres (4)

1. Allow replication or extension of chromosome ends.

2. Prevent chromosome ends from being ligated together.

3. Attach chromosomes to the nuclear lamina (envelope)

4. Protect chromosome ends from exonuclease attack

(5. Begin synapsis of chromosomes during meiosis)

b. Topoisomerase I in DNA replication (1)

Makes a cut in one of the two strands of DNA so the DNA can rotate and relieve tension caused by unwinding the DNA

c. DNA primase in DNA replication (1)

Produces an RNA primer to begin each Okazaki fragment on the discontinuous replicating strand

d. 7-methyl guanosine cap on mRNA (2)

1. Protects the 5’ end of the mRNA from exonuclease attack

2. Binds elongation factors to begin translation

e. Poly A sequence on mRNA (3)

1. Protects the 3’ end from exonuclease attack

2. Aids in exit of mRNA from the nucleus

3. Determines the t1/2 of the mRNA in the cytoplasm by a punch your ticket mechanism

(4. Aids in initiation of protein synthesis by interaction w the 5’ end
BSCI 421 Name

(20) 3. For two of the following four, explain how you would do experiments to do the following: (give the essential steps, including the enzymes or methods you would use)

a) Determine whether a sea urchin embryo is expressing a specific mRNA that you have

isolated from adult urchins.

I would make cDNAs from the adult mRNA with reverse transcriptase and DNA polymerase and incorporate radioactive labeled nucleotides. I would run the mRNAs from the embryo on an electrophoresis gel and blot it to nitrocellulose (Northern blot). Then I would denature the cDNA into single strands in NaOH, add the labeled cDNAs and let hybridize, cover with autoradiographic film, develop, and observe whether photographic grains are present over one of the mRNA bands.

b) Separate rat liver mitochondria from lysosomes, which are both about the same size and don’t separate well by velocity sedimentation.

I would homogenize the rat liver, do velocity sedimentation to isolate the fraction that contains both organelles. I would then place this fraction on a steep sucrose gradient (20-70%) and centrifuge at high speeds until the organelles reach equilibrium where the density of the particle = density of the solution. Mitochondria will be separated from lysosomes by this technique.

c) Produce a monoclonal antibody against a new protein you have isolated.

Inject the protein into a mouse (and wait for an immune response to develop). Remove the spleen cells and fuse spleen lymphocytes with a myeloma cell line lacking the enzyme (HGPRT) necessary to grow in HAT medium. Select hybrid cells by growing in culture in HAT medium. Clone individual hybrid cells in separate culture wells and test the solution over them for secreted antibodies against the original antigen (by an ELISA assay).

d) Determine whether the Origin of replication sequence and the membrane attachment site that separates replicated genomes are at the same point on a bacterial chromosome or whether they are separate points, (e.g., the replication forks are attached to the membrane as they separate).

I would insert the lac operon was next to the ori gene, and a

GFP gene in the lac I gene. Since lac I binds to lac O,

the I/GFP makes the gene fluorescent so it can be followed visually.

What I would see is the two newly replicated ori/lac regions moving apart

toward the poles in the plane of the membrane during DNA replication, so

the ORI is both the origin of replication and the membrane attachment site.

BSCI 421 Name ________________________________

Discussion questions: Write on one of the two discussion questions 4 or 5 for 20 points. (Leave blank or X out the one you are not writing on).

(20) 4. How does the synthesis, processing and function of eukaryotic messenger RNAs differ from eukaryotic ribosomal RNAs? (Contrast the two - you do not have to go into the details of the splicing mechanism)

Pre-mRNA (primary) transcripts are synthesized by RNA polymerase II,

processed by the addition of 7MG caps and poly A tails,

introns are removed, and the exons are spliced together.

Then the mRNA goes to the cytoplasm to be translated into polypeptide chains by ribosomes.

This synthesis and processing occurs at many different places throughout the nucleus.

Pre-rRNA transcripts for the large and small subunit rRNAs are synthesized by RNA polymerase I into one large, 45S RNA transcript.

7MG caps and poly A sequences are not put on rRNAs.

The 45S RNA is then cut into smaller pieces with “introns” removed but the cut ends are not spliced together.

The smaller piece (about 18S) binds a number of proteins and becomes the small ribosomal subunit.

The larger piece is split again into the 28S and 5.8S rRNAs.

A 5S rRNA transcribed from a separate gene and a large number of proteins add to these rRNAs to make the large ribosomal subunit.

The two subunits go to the cytoplasm independently and form a functional ribosome when they bind to a mRNA to translate it.

The synthesis and assembly of ribosomes occurs at a specific site or sites within the nucleus called nucleoli.

BSCI 421 Name ______________________________

(20) 5. Draw and discuss chromatin domains. What are they? What do they include? (Draw an active domain flanked by two inactive domains and label each component) What are the functions of each component of an active chromatin domain? How do domains change during activation of a previously inactive domain? How many ORIs does each domain contain and what is the significance of this?

Chromatin domains are large regions of chromatin that function and replicate as a unit.

They include two scaffold - association/attachment regions (SAR's) of DNA at each end to which scaffold-attachment proteins (SAPs) bind and attach domain ends to the protein scaffold of the chromosome. These serve as boundaries or insulators to prevent LCRs or enhancers from one domain from regulating genes in another domain.

Topoisomerase II molecules allow domains to expand by cutting both strands of one DNA molecule and passing the other through to resolve tangles.

A locus control region regulates whether a domain is open (expanded to euchromatin) or closed (heterochromatin) by modifying the nucleosomes (or chromatin structure) in the domain. This is probably by introducing chromatin remodeling proteins at this site.

One or more (an average of three) individual genes are individually regulated at their promoters and transcribe RNAs.

One origin of replication is in the center to replicate each domain as a unit.

The significance of this is that some domains replicate early and some late; early replicating domains will have the first opportunity to acquire transcription factors to turn the gene on and thus will have active genes.

(Draw a domain and label each component - see your notes and the .ppt fig I drew)