 |
 |
||
| Bioc 571 Midterm Exam Key | |||
Download PDF file
The following 16 questions are worth 5 points each.
1. What two factors contribute to the biochemical instability of RNA compared to DNA in laboratory research? Why is RNA instability a "good thing" in biological processes?
Biochemical instability is due to the presence of RNase enzymes on labware and human hands, and the fact that RNA is chemical unstable due to 2'-3' cyclic phosphate formation.
RNA instability is a good thing in biological processes because it facilitates gene regulation by ensuring that transcriptional initiation events are the rate-limiting step.
2. What is the biochemical basis for an increase in Tm of DNA in the presence of high [Na+]?
Na+ shields the negatively charged phosphate backbone which decreases repulsion between strands. Therefore more thermal energy is required to disrupt hydrogen boding between base pairs.
3. What is IPTG and how does it work to regulate gene expression in the lac system?
IPTG is a non-hydrolyzable analog of lactose (allactose) which binds to the lac repressor protein, resulting in de-repression (activation) of the lac operon (lac promoter).
4. Give an example of an antibiotic resistance gene and describe how it is used in applied molecular genetics?
The ampicillin-resistance gene (ampr) encodes the enzyme beta-lactamase which is secreted and hydrolyzes the antibiotic ampicillin. Ampr is used as a dominant selectable marker to detect plasmid replication in transformed E. coli cells.
5. What is a plausible explanation for how DNA transformation by chemicals and electroporation results in DNA uptake by transfected cells?
Transformation by chemicals and electroporation may cause a transient opening in membrane channels or pores which allows DNA to enter the cell.
6. What does the orientation-dependence of the M13 ori tell you about filamentous phage replication, and how is this different than lambda phage replication?
M13 replication uses the rolling circle mechanism and only one of the two strands is synthesized. Therefore, depending on the orientation (direction in terms of clockwise or counterclockwise) of the F1 ori in the phagemid, the product will only be of one strand or the other. Lambda phage replication is bi-directional and produces a double strand product.
7. What are two explanations for low intensity signals of positive plaques during the final purification step when screening a lambda phage genomic DNA library?
Low homology between two gene sequences; imperfect match means less signal due to loss of labeled hybrid in regions of non-homology.
Small stretch of 100% overlap as you would get with library walking, e.g., just the terminal 100 bp are complementary resulting in a weak signal.
8. Why would linker scanning NOT be a good mutagenesis strategy for investigating protein structure and function?
Because 2/3 of the ligated products would be out of frame ,and moreover, some base changes would create termination codons.
9. What is the difference between using classical genetics and "reverse" genetics in the context of yeast-based molecular genetic studies?
Classical genetics is based on an observed [mutant] phenotype that is then used to isolate the causative gene using linkage maps and physically mapping.
Reverse genetics is based on first identifying a segment of DNA thought to contain a gene of unknown function. A mutation is introduced into the gene and then the researcher looks for an observable mutant phenotype, e.g., developmental defect or response to environmental stimuli.
10. How could the EMSA assay be used to measure DNA binding affinities of a purified protein?
Titrate a competing unlabeled oligo against the labeled oligo to obtain a relative affinity value for the specific site. A high affinity DNA binding protein will require a large amount of unlabeled competing oligo before a decrease in EMSA signal is observed. Specificity of binding can be measured by competing with an unlabeled mutated (or non-specific) oligo.
11. Does the cDNA synthesis replacement strategy of Gubler and Hoffman result in the recovery of a cDNA product that includes the corresponding +1 nucleotide of the mRNA transcript? Explain.
No because the 5' terminus contains the RNA primer which cannot be replaced by cDNA.
12. Would blue/white screening distinguish between recombinants that contain cDNA inserts, and those that contain adaptor dimers, if the adaptor backbone were 12 nucleotides long? Explain.
Most likely because an adaptor dimer would be 24 nucleotides long which is 8 codons and would result in the synthesis of a functional alpha subunit, assuming there is no in-frame termination codon in the adaptor sequence. The reconstitution of beta-galactosidase as a result of no insert (vector religation) or an in-frame adaptor dimer would still be different than loss of alpha subunit due to most large cDNA inserts.
13. What is included in the hybridization solution to minimize differences in oligonucleotide Tm when screening a cDNA library with a degenerate oligonucleotide probe? Explain.
TMAC (tetramethylammonium chloride) which has a high density of positive charges and thus effectively stabilizes hybrid molecules independent of sequence composition.
14. List two criteria that must be met for a protein activity assay to be a viable strategy for cDNA library screening.
The desired gene must be expressed in the cell type used to construct the cDNA library.
A full-length, or at least functional, cDNA clone must exist in the library.
The protein activity must be encoded by a single polypeptide chain (single cDNA).
15. Why are both the Gal4-dependent His3 and LacZ reporter genes used in the yeast two hybrid screening strategy? Which is a genetic screen and which is a genetic selection?
The rate of false positives is very high in the yeast two hybrid screen and therefore it is important to have more than one indicator that a reconstituted transcription function exists in the cell (protein-protein interaction is occurring between the bait protein and target protein).
His3 is a genetic selection and LacZ is a genetic screen.
16. Why can't sodium hydroxide (NaOH) be used to denature RNA in an agarose gel that is being used for Northern Blot analysis?
Sodium hydroxide hydrolyzes RNA by catalyzing the formation of a 2' - 3' cyclic phosphate.
