BIO 510 Final Exam
Brief outline of answers
1. (10 pt) Match the following compound with the process or application.
A)Tryptophan-dependent repression of gene expression, B) horseradish
peroxidase substrate, C) mutagen, D) chromogenic reagent for alkaline
phosphatase reaction, E) negative selection against URA3 + yeast
E
A
C
D
B
2. (2 pt) Compared with the pTZ19u plasmid used in the first half of the semester,
pBR322 has a (lower) intracellular copy number due to the presence of (circle all
correct answers):
ROP gene
3. (3 pt) The plasmid pBR322 is an example of a (restricted host range) plasmid.
Describe how pBR322 regulates its intracellular copy number by modulating
replication. Col EI-type replication with 555 nt RNA II being an RNase Hdependent plasmid-encoded replication primer and RNA I an antisense 108 nt
negative regulator that blocks RNase H cleavage & replication. Rop stabilizes the
RNA-RNA hybrid.
4. (5 pt) Investigator James Goofup mistakenly used a rabbit anti-yeast tubulin
primary antibody to probe a western blot to identify a TAP-tagged ubiquitin
hydrolyase protein. After washing, he added a goat anti-rabbit-alkaline
phosphatase secondary antibody. When he used the BCIP/NBT reagents he
detected TWO bands ONE of which was later confirmed was the correct yeast
ubiquitin hydrolyase. Provide an explanation why this experiment “worked” even
though an incorrect primary antibody was used (that is, the primary antibody was
directed against a protein that had no antigenic similarity to ubiquitin hydrolyase).
The primary antibody was irrelevant as the secondary antibody is able to bind
directly to the protein A segment of the TAP epitope present on the ubiquitin
hydrolyase.
5. (5 pt) Compare and contrast the Roche 454 and Ion Torrent sequencing
methodologies. Describe the sample preparation, the sequencing reaction, and the
means of “base calling” (that is, how is it decided which base is added). See lab
Powerpoint slides describing the emulsion chemistry for DNA-bound beads
followed by pyrophosphate chemistry. See embedded Ion Torrent link where
polymerization results in a hydrogen ion release that is detected as a pH change.
6. (5 pt) What is the Nanostring technology used for? How does this technique
work? Describe the details of sample analysis and the underlying basis of this
technology. See PowerPoint lecture with the embedded web link. In essence, this
is a machine for monitoring specific mRNA abundance, requires two substratespecific oligonucleotides, one biotin-substituted for mRNA capture, the second
containing an ordered array of florescent tags for mRNA identification.
7. (10 pt) 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, D, E, I, G, H) Negative interactions (B, C, F). 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 (i.e., in lab, but not in this
exam, we considered the boxes to be the deleted region).
Interpret these results – specifically, tell which peptide segments of Pxr1 are
sufficient for interaction with actin. Two non-contiguous regions of Pxr1 interact with
Prp43. These are defined by amino acids 100 to 150 and amino acids 225-271.
8. (7.5 pt) You want to test the hypothesis that the protein encoded by the HSF1
gene interacts with the Cyc8 protein. You successfully and properly clone HSF1
into the pAS2 yeast two-hybrid vector and successfully and properly clone CYC8
into the pACT yeast two-hybrid vector. You include the empty vectors (that is,
no insert) of pAS2 and pACT as negative controls. The following results were
obtained:
Y 2-Hybrid
constructs
Transactivation
of GAL1-HIS3
(i.e., His+)
Transactivation
of GAL2-ADE2
(i.e., Ade+)
pAS2-HSF1
+pACT-CYC8
pAS2-empty
+pACT-CYC8
pAS2-HSF1
+pACT-empty
pAS2-CYC8
+ pACT-HSF1
pAS2-CYC8
+pACT-empty
pAS2-empty
+pACT-HSF1
pAS2-empty
+pACT-empty
+++
+++
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Please note that the correct yeast host strain was used that included two legitimate
reporter genes for transactivation (GAL1-HIS3, GAL2-ADE2). There were no mistakes in
cloning (that is, the plasmids are constructed appropriately) or in the yeast transformation
and there was no contamination on the plates and no errors in scoring the results. That is,
the results are 100% correct as presented and can be reproduced by any investigator.
a. Based on this data, do you conclude that the Hsf1 and Cyc8 proteins interact
or not? In your response be clear and provide a full explanation of the basis
for your decision.
b. How do you interpret the pAS2-HSF1 + pACT-empty results? Provide a
scientific explanation for how this might occur – remember, no errors of
experimental design, execution or interpretation occurred.
No, it appears that these do not interact. The HSF1 segment is itself capable of
stimulating transcription when bound to the DNA binding domain of Gal4 (autoactivation). However, when put into the activation domain vector, it cannot interact with
Cyc8 indicating that these two proteins do not bind one another)
9. (5.0 pt) The Protoscript M-MuLV Taq RT-PCR kit was used for cDNA
synthesis in the lab and the manual accompanying this kit assigned as required
reading. Three different oligonucleotide primers were described for first strand
synthesis,
a. an anchored oligo dT – works well for 3’ ends and full-length cDNAs; note
that the anchored feature diminishes promiscuous amplifications from
internal A-rich sequences.
b. a random prime mixture – provides broad coverage even for very long
transcripts that are difficult to amplify with “a”; also good for RNAs that
lack poly A tails.
c. a gene-specific oligonucleotide directed at the transcript of interest – most
specific as this will amplify only the transcript of interest. Directs all
resources (enzyme & nucleotides) towards the gene of interest and may
give greatest yield. Good for direct cloning of specific mRNAs.
Describe the benefits and limitations for use of a, b or c for first strand synthesis. As part
of your answer, state when you would choose one primer over another on this list.
10. (8 pt) The XL1-Red mutagenesis approach was used in the lab and the manual
accompanying this kit assigned as required reading. Three different mutations are
present in the XL1 background, The mutS, mutD and mutT .
a. Describe what the specific natural function for each of these genes (i.e., mutS, mutD
and mutT ) and discuss how the loss of each function can increase mutagenesis of
plasmid DNA. These include The mutS ( required for DNA mismatch repair
pathway), mutD (deficient in the 3´- to 5´- proofreading exonuclease of DNA
polymerase III) and mutT (this mutant is unable to hydrolyze 8-oxo-dGTP, produced
by natural oxidative damage to DNA and modifying the base to allow
basepairing with all nucleotides – but especially increasing the occurrence of
dG-dA mismatches.
b. When plated, the XL1-Red mutator strain causes a wide range of colony sizes (even
when no plasmid is present). Assuming that there is no contamination, speculate why
variable colony sizes are observed.
i.
If you isolate 10 large XL1-Red colonies form a plate, propagate each
independently for 100 generations and then replate, what do you expect
to see in terms of % large/small colonies compared with the original 10
colonies used for propagation? Since small colonies are defined by cells
that divide more slowly due to mutation. As the XL1-red cells randomly
mutate the DNA one expects both large and small colonies to arise after
propagation – with variability in the % small between the cultures
depending on when the mutation arose during culture.
ii.
If you isolate 10 small XL1-Red colonies form a plate, propagate each
independently for 100 generations and then replate, what do you expect
to see in terms of large/small colonies compared with the original 10
colonies selected for propagation? Since the cultures were originally
mutant and will gain further mutations one expects most will remain
“small”. However, extragenic suppressor mutations (or direct reversion
of the original mutation) can occur and under selective pressure such as
this some “large” colonies might be observed (as faster dividing cells
become enriched).
11. (5.0 pt) The DX1R transcript is 300 nts in length but processed into multiple
mRNA isoforms, including the joining of exons: 1-2-3-4-5, 1-2-4-5, 1-2-5 and
very rarely, 1-5.
You have access to Illumina sequencing, Roach 454 sequencing and to Affymetrix tiling
array technology. Assume that exons 1, 3 and 5 are each 75 bp in length, exon 2 is 50 bp
and exon 4 is 25 bp.
- Which method would be best to determine the relative fraction of all
mature mRNAs? That is, what % are made as 1-2-3-4-5?, % 1-2-4-5?, %
1-2-5? and % 1-5?.Justify your answer by stating why this method is best.
This is really a “read length” issue and the Roach 454 gives the longest reads of the
three methodologies.
12. (7.5 pt) Explain how ONE of the following genetic approaches can be used to
identify a novel gene involved in yeast pre-mRNA splicing. A) Dosage
suppression, B) extragenic suppression, or C) Synthetic lethality. See lab notes –
dosage suppression works by increased gene product abundance, usually plasmid
based where overexpression is achieved by the use of a strong promoter or highcopy number plasmid. Extragenic suppression generally done by mutating a
genome of a cell bearing the primary mutation to identify second-site “hits”
(mutations) that allow the double mutant to grow better than cells harboring only
the first mutation. Synthetic lethality involves the combination of mutations to
identify those that are independently viable but lethal when combined. In all cases
you need to describe how the gene would be cloned (A –already done; B – could
be done by a plasmid library screen – the source of the library depends upon
whether the suppressor is dominant or recessive, C, direct cloning with a plasmid
library to reverse the synthetic lethality). Finally, to describe how you would
create a mutant allele in the recovered gene & test for function (many ways
possible).
In your answer be sure to describe each of the technical/experimental steps needed to i)
identify the gene, ii) clone the gene, and iii) confirm that the cloned gene is actually
involved in pre-mRNA splicing. Describe all yeast strains that are needed, including any
genetic markers required for plasmid selection or phenotypic screening. Provide clear
details on each of the experimental steps – for instance, if you need to use a mutant
construct, describe how that construct would be made. NOTE: answer for A, B, or C –
do not do more than one, no extra credit will be given.
13. (5.0 pt) Describe how TAP-tagging of proteins can be a) combined with mass
spectroscopy to identify multi-subunit protein complexes within cells and b)
define the subcellular location of individual proteins. Tandem affinity purification
is a very effective way to recover protein complexes - TAP-tag one gene, express
it in the cell type of interest, recover by sequential IgG agarose-Calmodulin
agarose selection, the release the peptides by EGTA. Run a 2-D gel for MALDITOF to identify the associated proteins one-by-one or do ESI using a MS/MS (or
LC/MS) approach to define all associated peptides in the pooled sample.
14. (5 pt) What is meant by “paired-end” DNA sequencing? Describe how this is
done. Look in PowerPoint slides – essentially, taking a DNA fragment with
defined ends containing an “infrequent cutter” endonuclease site in which the
enzyme cleaves 20-30 downstream of the recognition motif. Cut with the enzyme
to release internal sequence, then join the ends. This allows you to “hop” along
a sequence to read nearby but not adjacent sequence – this is especially helpful
for aligning sequence in regions of repetitive DNA.
15. (7.5 total) How do the clones recovered from a cDNA library differ in structure
from those recovered from a genomic DNA library? What sorts of chromosomal
sequences do you expect to be found exclusively in the genomic DNA library?
What specific sequence commonly associated with mRNA will only be found in
the cDNA library? Genomic – non-transcribed DNA (intergenic regions,
centromeres, telomeres), promoter regions of genes, sequences downstream of the
polyA site, intron sequences. cDNA – none of the above but the cDNA will have
the non-encoded polyA tail.
What factors do you consider when estimating the number of clones that need to
be screened to find a gene of interest in 1) a cDNA library (mRNA abundance or
enrichment) and 2) a genomic DNA library (genome complexity and length of
DNA insert)?
16. (12 pts) Define a) inclusion bodies (insoluble precipitates of a foreign protein
when expressed in bacteria), b) TY1 (yeast retrotransposon), c) 8-oxo-dGTP
(natural oxidative damage to dGTP –alters basepairing potential), d) sonicator (machine
used to break DNA, lyse cells or solubilize protein mixtures by converting electrical
stimulation into oscillating mechanical vibrations that create microscopic shock waves
that do the “work” of the sonicator, e) 3-aminotrizole (chemical inhibitor of the
URA3 enzyme, imidazoleglycerol-phosphate dehydratase, f) random prime
labeling (DNA labeling by the annealing of a population of oligonucleotides of
random sequence to a single-stranded target followed by extension with DNA
polymerase in the presence of a radiolabeled (biotinylated, etc) nucleotide.
Assumption is that within the complex mixture of short oligonucleotides there will
be a subset specific for the target DNA).