Final exam average grade was 70. No curve was applied to the exam grade but 3 points
were added to everyone’s final grade (which results in a much bigger positive impact than
adjusting just the final).
Bio 510 Final Exam Fall 2014
NAME_________________________________
1. (6 pts) Which of the following DNA elements are most often enriched in A) a genomic DNA
library or B) an oligo dT primed cDNA library, C) both A and B, or D) neither A nor B, “to each
of the following:
A__ intact full centromere
B___ poly A tail
A___ introns
B (or C) protein coding sequence
A functional DNA replication origin
A tRNA coding sequence
2. (10 pts) Which of the following would be useful for (put letter alongside protein or process:
1) ___A___developing a western blot image, 2) __C__use with EcoRI methylase, 3)___D__
elution of peptides from a streptavidin column, 4) __E___solubilizing E. coli, : 5) ___B___intein
cleavage
A)
B)
C)
D)
E)
3. (4 pts) You complete an Ion Torrent-based sequencing study of cDNA prepared by random
prime labeling. You find evidence for anti-sense RNA transcription across the yeast CYC1 gene.
This means that you find examples of RNA that has the opposite polarity of the natural CYC1
mRNA. Design experiments that:
a. describes an alternate approach to demonstrate that both anti-sense RNA really exists in the
cellular RNA population. This could be done by northern blot with single-stranded riboprobes or
using an rtPCR approach with a strand-specific oligonucleotide priming first strand synthesis.
b allows you to determine whether or not the antisense RNA is polyadenylated. Hybridize the
RNA with oligo dT then treat with RNaseH. If there is a poly A tail, the mRNA will be shifted
in a northern blot by the length of the poly A tail. Alternatively, use an rtPCR approach with
first strand synthesis is primed by oligo dT.
For each question, clearly explain the methodology involved, how probes will be prepared,
data gathered and results interpreted.
4. (4 pts – two part question) a) Describe the biological or biochemical properties of each of the
following and b) tell how each was used our BIO 510 lab experiments?
a. nitrous acid – chemical mutagen that deaminates cytosine and adenine resulting in
GC->AT and AT->GC transitions; used this for mutagenesis of lacZ within single
stranded pTZ18u
b. 5 fluoroorotic acid –The toxic anti-metabolite, 5-fluorouracil, is produced from 5
FOA in URA3 + background. We used this in a plasmid shuffle experiment to test the
function of certain mutant genes.
c. BCIP/NBT Chromogenic substrate of alkaline phosphatase (AP); used to detect TAPtagged proteins bound with the AP-conjugated secondary antibody
d. Reverse transcriptase – RNA-dependent DNA polymerase used for first strand
synthesis in our cDNA preparation.
5. (12 pts) Using the following hybridization data, determine the location and direction of
transcription for the four RNA transcripts. GIVEN: Assume that no more than one transcript is
present in any interval defined by two adjacent restriction sites (that is, BamH1 to Pst1, Pst1 to
Tsp1, Tsp1 to Msp2 etc.) and that no transcripts extend into an adjacent restriction fragment.
Put an arrow head at the 3’ end of the transcript (5’ -> 3’) and be sure to indicate clearly by a
solid line labeled with a transcript length where each transcript resides and label its size.
Single stranded Probe (5’->3’) Hybridizing Bands on Blot (all in kb)
BamH1-> Cla1
0.8 kb, 3.1 kb
Msp2-> Pst1
1.7 kb
BamH1-> Msp2
Cla1-> BamH1
Pst1->Gas1
Tsp1-> BamH1
Pst1->Msp2
0
1.0 kb, 1.7 kb
0.8
1.0 kb
0
The restriction sites are indicated below by the first letter only, e.g., B = BamH1, P=Pst1 etc.
BamH1 1.0-> Pst1 Tsp1 1.7->
Msp2 <-0.8 Gas1 <-3.1
Cla1
6. (2 pts) How do monoclonal antibodies differ from polyclonal antibodies? Would a
monoclonal or polyclonal antibody likely be better for the selective identification of one member
of a group of related proteins? In your response, provide a rationale based on the properties of
monoclonal and polyclonal antibodies that would make you choose one type over the other for
this particular application. Monoclonal antibodies are produced from a single clonal hybridoma
(transformed B cell), each monoclonal antibody preparation is therefore composed of identical
immunoglobulins that bind a single epitope of the target. Polyclonal antibodies are mixed
populations of antibodies composed of multiple biochemically distinct species that typically
target multiple regions on the target. Given the specificity, the monoclonal antibody approach
gives you the best chance of distinguishing one protein from another in a family member from
another in a group of related proteins.
7. (3 pts) What does the term “eipgenetics” mean? This refers to helitable forms of gene
regulation that rely on features in addition to the DNA sequence to modulate the expression of
genes. This can include, for instance, DNA methylation or a variety of histone methylations
(e.g., phosphorylation, methylation, acetylation), regulation by miRNA or other trans-acting
modulators.
8. (2 pts) MALDI-TOF and ESI-MS/MS are two alternative mass spectroscopy approaches
applied to proteomic studies. (MALDI-TOFuses laser excitation to ionize peptides. (ESIMS/MS circle one) generally provides more detailed peptide information resulting from greater
numbers of peptide ionization states.
9. (2 pts) Mass spectra are generally graphed as relative intensity (Y axis) against the m/z ratio
(X axis). What values do the “m” and “z” terms define? Mass(m) and charge (z)
10.(2 pts) Restricted plasmids (e.g., Col E1 derived) have limited bacterial host while
promiscuous or broad host range plasmids (e.g., RSF1010) can replicated in a wider selection
of bacterial hosts. What natural biological differences in plasmid biology account for this
difference in host range? The restricted plasmids rely to a greater extent on host factors for
plasmid replication and/or segregation while the broad host range plasmids themselves encode
factors needed for plasmid propagation.
11. (3 pts) Describe two ways in which Affymetrix arrays and printed cDNA arrays differ in
preparation or use (for instance, when you want to measure mRNA differences between healthy
and cancerous tissues). The Affymetrix arrays contain short (~21 nt) oligonucleotide probes
synthesized on the slide by lithography. The printed arrays can be prepared in several ways,
including covalently binding long (50 to several hundred bp) DNA PCR fragments to the glass
slide. The Affymetrix slides are hybridized with a single cRNA preparation made from either
the control or experimental system while the cDNA arrays are often simultaneously hybridized
by complementary cDNA samples labeled with two different photofluors for the control (e.g.,
Cy3) and experimental (e.g., Cy5) samples.
12. (3 pts) What does “paired-end sequencing” mean? How is this done? See currently posed
Powerpoint slide #167. What common problem encountered during genomic sequencing of
complex organisms does the paired end sequencing strategy help alleviate? The difficulty of
aligning sequence through runs of highly repetitive DNA
13. (2 pts) Which of the following sequencing strategies involved the incorporation of a chainterminating modified dNTP (circle all that apply)?
c. Illumina sequencing
d. Classical “Sanger” sequencing
14. (4 pts) Promoter fusions and protein (or peptide) fusions are commonly used in molecular
biology. Discuss one advantage and one disadvantage for the use of promoter fusions and one
advantage and one disadvantage for the use protein fusions. See current Powerpoint slide #124
15. (4 pts) What is the specific molecular target of each of the following affinity matrixes used
for protein purification.
Streptavidin-agarose biotin
Chitin-agarose
chitin binding domain peptide
IgG-agarose
protein A protein fusion
Nickel-NTA-agarose His6-protein fusion
16. (5 pts) What is meant by the term “nested” PCR? Describe in detail how such an experiment
is conducted. As part of your response add oligos on the figure below assuming that you want to
amplify the CRX1 gene. This is a technique of sequential amplification designed to avoid offtarget amplification when using highly complex template DNAs (e.g., human genome). To do
this, conduct the first ~ rounds of amplification using distal primers 1 & 2 following by ~20
rounds of amplification using primers 3 & 4.
__________________________///////////////////__________________________________
----------------------1
3
4
2
17. (10 pts) Design an experiment to identify genes acting in the process of pre-mRNA splicing
using either a 1) synthetic lethal, 2) dosage suppression, or 3) extragenic suppression approach
(select only ONE approach). In your answer, you must clearly:
1) describe all mutations in any chromosomal or plasmid-based genes used in this study.
The description should include the characteristic of the mutant allele (e.g., temperature sensitive,
lethal, fully functional, partial loss of activity, etc.)
2) clearly state what culture conditions (e.g., media type & temperatures) will be used
3) state how the wildtype alleles of any mutants found in your study will be cloned
4) state how you will test whether or not the genes found in your study actually encode
proteins important for pre-mRNA splicing. Here, it will be important to clearly describe the
assay that you will use to learn if the identified gene is required for splicing or not. See posted
on the class website:



Extragenic suppression paper
Dosage suppression paper
Synthetic lethality paper
18. You have performed a yeast two hybrid analysis to map the location of Pxr1 interaction with
actin. You get the following results: Positive interactions: A, C, D, H) Negative interactions (B,
E, F, G, I). NOTE: In the diagram below, the boxes represent the fragments that ARE
INCLUDED in the yeast two hybrid construct tested. For instance, construct “A” tested the
whole protein while construct “B” contained ONLY amino acids 25-70 fused to the Gal4. This
is opposite of how the diagram was used for our lab experiment (i.e., in lab, but not in this
exam, we considered the boxes to be the deleted region). Assume that the binding domain is
limited to one segment only (i.e.,1-25, 25-70, 70-101, 101-150, 150-226, or 226-271)
Based on these observations,
a. (2 pts) Which peptide segment of Pxr1 appears essential for interaction with actin? The
peptide 70-101.
b. (3 pts) Design an experiment to test whether the peptide segment you list in part “a” is
sufficient for this interaction. Be clear in what genetic constructs you would make and how each
would be scored for interaction. Redo the yeast 2 hybrid assay with the Pxr1 (70-101) against
actin. If your prediction is correct, this peptide should be sufficient for interaction. If it does not
show a positive Y2H response, then it is necessary, but not sufficient.
19. (2 pts) Define the features of plagiarism as this relates to any scientific writing. That is, what
specific things would be present in a document that would demonstrate that the document is
plagiarized? See your lab manual page 11.
20. (5 pts) Starting with one double-stranded DNA template (shown below), draw all the
products of 1, 2, and 3 cycles of PCR using the primers indicated. In drawing out the products
be sure to distinguish between ends that incorporate the primer and those that found in the
original DNA but excluded by PCR. For instance, to show this, you can use wiggly lines to show
primer ends.
How many fully double stranded DNA molecules that terminate at both ends with the primer
sequence will be present after:
1 round of PCR amplification? 0
2 rounds of PCR amplification? 0
3 rounds of PCR amplification 2
21. (1 pt) Nitrous acid (HNO2), which acts as a mutagen by conversion of a ring amine group of adenine
and/or cytosine to an ether group, thus altering their base pairing. We used this for the random chemical
mutagenesis of pTZ18u DNA.
22. (3 pts)When monitoring the effectiveness of nitrous acid (NA) mutagenesis, we scored the
number of ampicillin resistant, lacZ deficient colonies on our control (-NA) and experimental
(+NA) plates. Does the % white colonies tell you the percentage of cells bearing a mutation in
the lacZ gene? Write a full response and tell why or why not you believe that this is true. If one
assumes that each colony has a single population of identical plasmids (good assumption), the
number of white colonies defines the number of lacZ inactivating mutations (that is, mutations
that fully block β galactosidase activity). However, lots of base pair changes will tolerated and
not result in changed amino acids or, even when the amino acid changes, not fully inactivate
lacZ. So, even leaving aside the idea that white colonies might arise for other reasons (e.g.,
mutations that make the cells improbable to IPTG), the white colonies will underestimate the
number of lacZ mutations.
23. (2 pts) What is the natural biological role of an intein? That is, what purpose does the intein
serve in cellular biochemistry outside of the research lab? The intein is an internal portion of a
protein capable of auto-excising (protein self-cleavage and ligation). This feature is used for
regulating protein activity, such as in the DNA gyrase protein whose gene was used for the
preparation of the NEB pTXB1 vector. Here intein excision is needed for gyrase activity.
24. (2 pts) Describe the mechanism by which ATP promotes DNA ligation in vitro. Be specific
about the chemical interactions involving ATP. See currently posted Powerpoint slide #84.
25. (2 pts). Offer two specific suggestions on how we can improve the BIO 510 course in future
offerings. The suggestions can be for changes in course content or organization but should retain
the current class hours and credit. Credit will be given only for positive, thoughtful responses.
Lots of very valuable comments were offered – thanks.