BIOLOGY 416
NAME:_____________________
MOLECULAR VIROLOGY
EXAM #1
FRIDAY FEB. 14, 2003
(A) Answer each of the following questions in the space provided.
(B) Be sure to write legibly.
A.
Answer the following short answer questions. (46 POINTS TOTAL)
1. Restriction enzymes are extensively used for cloning. Where do we isolate restriction enzymes
from and what purpose do they serve in these organisms? (6 points).
Isolated from bacteria.
Serves as a defense mechanism against foreign DNA. Restriction enzymes digest foreign
DNA such as those from bacteriophages.
2. Describe in detail a quantitative assay that can be used to measure the amount of virus in a
sample. Provide enough information for the reader to perform such an assay (8 points).
Do a plaque assay: titrate 10x serial dilutions of virus on a susceptible monolayer of cells;
following addition of virus, cells overlaid with agar and incubated. Several days later
plaques are visualized by staining live cells and observing 'holes' of dead cells, count
plaques.
3. List three features that are found in most eukaryotic genes (6 points).
Promoter, introns, exons, start sites and termination sites.
4. Virus X produces a replication protein in both brain and kidney cells, but at different levels.
Describe how you would examine the replication protein of this virus in these cells. What
reagents would you need (8 points)?
Do a Western blot on cytoplasmic extracts of both brain and kidney cells that have been
infected with the virus. You will need to run an SDS-PAGE gel of the cells extracts
which will then be transferred to a nitrocellulose membrane. The membrane will then
be blocked with nonspecific protein such as milk and then probed with a primary antibody
that is specific for the replication protein. A secondary antibody with some kind of label
(chemiluminescent or radioactive) will be used to detect the primary antibody. You will
need film which can be exposed to the nitrocellulose blot so that you can visualize the
protein bands.
5. The genome of a (+) strand RNA virus is said to be infectious. Explain? (4 points).
The genome of a (+) strand RNA virus directly acts as an mRNA. When introduced into
cells it will use the cells translation machinery to produce viral proteins which in turn
will replicate the genome and result in the production of virus particles. Thus, the RNA
genome is itself infectious.
6. Explain or diagram how you would introduce a mutation into a fragment of DNA by PCR (6
points).
This is from your notes. (see page 43)
7. Describe the important physical features of a defective-interfering RNA. Explain why it is
called a defective-interfering RNA (8 points).
Contains cis-acting sequences: plus strand and minus strand promoter and packaging
sequences. Since it lacks most of the protein coding sequences it cannot replicate by
itself (defective) and must rely on a helper or wild type virus. Since it utilizes viral
proteins for replication it competes with the wild type RNA and therefore 'interferes' with
wild type replication. Being smaller and having the same signals it is replicated at
higher levels than the wild type.
B.
Answer the following TRUE (T) OR FALSE (F). ( 3 points each, 30 POINTS TOTAL)
___F___ 8. RNA-dependent RNA polymerases are found in the nucleus of most eukaryotic
cells.
___F___ 9. Recombination in RNA viruses via template switching occurs prior to RNA
replication.
___F___ 10. All mutations produce an observable phenotype
___F___ 11. The DNA coding strand serves as the template for synthesis of mRNA.
___F___ 12. In a persistent infection, infectious virus is never released from the cells.
___F___ 13. Penetration of viruses or their genomes into the host cell is an energy-independent
event.
___F___ 14. The genome of a minus-strand RNA virus is sufficient to initiate an infection
when introduced by itself into a susceptible cell.
___F___ 15
BIO 416 Exam 1
Complementation between two distinct parental viruses results in progeny virions
containing new genotypes.
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___T___ 16. Reassortment of influenza genome segments can occur when two genetically
different viruses infect the same cell.
___F/T___ 17. The high error frequency of DNA viral genomes can be attributed to the lack of
proof-reading by their DNA polymerases.
C.
Answer the following problem solving question (24 points)
18. As the senior virologist at Special Pathogens Branch of the CDC, you are given a very small
sample of a novel virus that produced encephalitis in humans in the southwestern United
States. Your job is to characterize the virus and assign it into an appropriate virus family.
Describe how you would carry this out and pay attention to detail. Make sure that you
describe how you would (a) physically analyze the virus and (b) genetically analyze the
virus.
a)
1.
2.
3.
Amplify virus in cell culture or animal host.
Purify it.
Examine i) particle, ii) proteins, and iii) nucleic acid.
i)
particle: determine if it has envelope (detergent treatment) and look at it
by EM.
ii) Analyze protein components by gel
iii) Determine RNA/DNA, ss/ds and linear or closed circular using specific
nucleases. Also determine how many fragments per genome by analysis on gel.
b)
Prepare cDNA. 1. Sequence: Determine gene order and nature of proteins.
2. Probe function by mutagenesis.
These steps will help you determine the family to which this virus belongs.
BIO 416 Exam 1
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