Deck 9: Tools for Analyzing Gene Expression

Question 1

During a transient transfection assay

A) DNA is introduced into a eukaryotic cell and remains extrachromosomal.
B) DNA is introduced into a eukaryotic cell and is stably integrated into a chromosome.
C) DNA is introduced into bacterial cells for amplification.
D) DNA is taken up and packaged in viral capsids.

Answer 1

DNA is introduced into a eukaryotic cell and remains extrachromosomal.

Question 2

You want to study regulatory regions that control expression of a gene whose protein product is difficult to measure quantitatively. What method should you use to analyze gene expression?

A) Western blot with an antibody specific for the protein of interest
B) Reporter gene assay
C) DNAse I footprinting
D) Coimmunoprecipitation assay

Answer 2

Reporter gene assay

Question 3

The technique of in situ hybridization gives information about:

A) which genes are being transcribed in a particular cell
B) which genes are present in a particular cell
C) which proteins are being degraded in a particular cell
D) which enzymes are active in a particular cell

Answer 3

which genes are being transcribed in a particular cell

Question 4

Western blotting is the process that is used to identify

A) DNA
B) RNA
C) protein
D) DNA-protein interactions

Question 5

Northern blotting is the process that is used to identify

A) DNA
B) RNA
C) protein
D) DNA-protein interactions

Question 6

Southern blotting is the process that is used to identify

A) DNA
B) RNA
C) protein
D) DNA-protein interactions

Question 7

To map the start of transcription for a given mRNA, the most useful method would be

A) RNase protection assay
B) RT-PCR
C) Northern blot
D) in situ hybridization

Question 8

A coimmunoprecipitation assay is a process that is used to identify

A) protein structure
B) transcriptional activity
C) DNA-protein interactions
D) protein-protein interactions

Question 9

A chromatin immunoprecipitation assay is a process that is used to identify

A) protein structure
B) transcriptional activity
C) DNA-protein interactions
D) protein-protein interactions

Question 10

You have received from a scientific collaborator some transgenic mice that have had the gene encoding the BRCA1 gene knocked out. Which method would help you to confirm the genotype of these mice most directly?

A) Southern blot
B) Northern blot
C) Western blot
D) Chromatin immunoprecipitation

Question 11

You are interested in an uncharacterized gene that contains an interesting combination of predicted protein domains. To determine in which mouse tissues it is expressed, the best method is

A) DNase I footprinting
B) transient transfection assay
C) Northern blot
D) coimmunoprecipitation

Question 12

You have identified a specific region in a virus that you suspect is bound by the host transcription factor Sp1. Which of the following methods could you use to test your hypothesis?

A) chromatin immunoprecipitation
B) electromobility shift assay
C) DNase I footprinting
D) all of the above

Question 13

Which of the following reporter genes allows one to visualize a protein in living cells?

A) beta-galactosidase ( $\beta$ -Gal)
B) green fluorescent protein (GFP)
C) chloramphenicol acetyltransferase (CAT)
D) luciferase (Luc)

Question 14

To analyze whether a putative transcriptional enhancer activates transcription, one could use which of the following approaches?

A) attach the enhancer to a reporter gene in a recombinant construct; transiently transfect the construct into cells
B) attach a histidine tag to the enhancer sequence in a recombinant construct; purify the complex using nickel-nitrilotriacetic acid (Ni-NTA) resin
C) co-transfect the enhancer sequence with a plasmid encoding RNA polymerase; perform in vitro transcription
D) use in vitro mutagenesis to disrupt the enhancer; immunoprecipitate the enhancer

Question 15

Which of the following is not a property of green fluorescent protein (GFP)?

A) GFP can be detected directly in living cells.
B) GFP requires addition of a cofactor for light production.
C) GFP contains an intrinsic peptide fluorophore.
D) Following excitation with ultraviolet light, GFP emits green light.

Question 16

You are analyzing the tissue-specific expression of a poorly characterized gene in mice. Which type of gene would serve as the best positive control for your experiment?

A) a known gene that exhibits temporal expression
B) a known gene that exhibits spatial expression
C) a known gene that exhibits constitutive expression
D) another poorly characterized or unknown gene

Question 17

Production of recombinant proteins in bacteria involves the cloning of the cDNA encoding the desired protein into an "expression vector" that contains a(n)

A) promoter
B) enhancer
C) intron
D) start codon

Question 18

Which method would best help you to identify specific amino acid residues that are critical for the function of a protein?

A) promoter bashing
B) yeast two-hybrid assay
C) alanine scanning mutagenesis
D) quantitative PCR

Question 19

Which component is not required for in vitro translation of a recombinant protein?

A) T7 RNA polymerase
B) amino acids
C) rabbit reticulocyte lysate
D) DNA polymerase

Question 20

The distance moved by a protein into a gel is inversely proportional to the

A) logarithm of molecular charge
B) logarithm of molecular weight
C) logarithm of molecular shape
D) Both A and C

Question 21

At its isoelectric point

A) a protein has a positive charge
B) a protein has a negative charge
C) a protein has no net charge
D) whether a protein has a positive or negative charge depends on the particular protein

Question 22

During the process of two-dimensional PAGE

A) the first dimension separates proteins by isoelectric point and the second dimensionseparates them by size.
B) the first dimension separates proteins by size and the second dimension separates themby isoelectric point.
C) the first dimension separates proteins by size and the second dimensionseparates them by shape.
D) the first dimension separates proteins by shape and the second dimension separates bysize.

Question 23

In an indirect immunofluorescence assay, which component is fluorescently labeled?

A) The antigen.
B) The primary antibody.
C) The secondary antibody.
D) both B and C

Question 24

Morpholino oligonucleotides have proved useful for

A) RNA interference (RNAi)
B) antisense-mediated inhibition of gene expression
C) reverse transcriptase-PCR
D) RNase protection assays

Question 25

Double-stranded RNAs are processed by the enzyme _______ to form small interfering RNAs (siRNAs). One strand of the siRNA is loaded into a silencing complex named _________. The siRNAs target mRNA for ___________ .

A) RISC, Dicer, degradation
B) Dicer, RISC, degradation
C) Dicer, RISC, splicing
D) RISC, Dicer, spicing

Question 26

You want to analyze the structure of a large protein complex at extremely high magnification. What method would you use?

A) confocal microscopy
B) fluorescence microscopy
C) cryoelectron microscopy
D) scanning electron microscopy

Question 27

You are testing protein X and protein Y in a yeast two-hybrid assay that uses lacZ as a reporter gene. Production of a blue product signifies that:

A) protein X interacts with protein Y
B) protein X does not interact with protein Y
C) protein X is required for production of protein Y
D) protein Y is required for production of protein X

Question 28

Which of the following has not been used as a model organism in molecular biology research?

A) Danio rerio
B) Caenorhabditis elegans
C) Arabidopsis
D) Homo sapiens

Question 29

Which of the following is a key advantage of NMR spectroscopy over X-ray crystallography?

A) NMR spectroscopy can accommodate larger sample sizes
B) NMR spectroscopy can be under dynamic conditions
C) NMR spectroscopy provides higher resolution data
D) Only NMR spectroscopy can be used to study DNA-protein complexes at high resolution

Question 30

A nonscientist friend of yours asks how findings in a worm or fly can be relevant to human biology. Explain to your friend the importance of model organisms in molecular biology research.

Question 31

Explain the information gained from an in situ analysis differs from that gained from an in vitro analysis, using protein or RNA expression as an example to illustrate your answer.

Question 32

Compare and contrast transient and stable transfection assays.

Question 33

Why is green fluorescent protein widely used as a reporter gene for investigation of tissue-specific gene expression and cellular localization or proteins?

Question 34

Describe the structure of green fluorescent protein and discuss how this structure relates to the function of the protein.

Question 35

Diagram a cotransfection assay. What information can be obtained from this type of assay?

Question 36

Describe the use of a vector that produces fusion proteins with glutathione-S-transferase (GST) at one end. Show the protein purification scheme to illustrate the advantage of the GST tag.

Question 37

You want to generate "optical sections" of a specimen. To this end, would you use conventional fluorescence microscopy or confocal microscopy? Explain your answer.

Question 38

What kinds of information can be obtained from a Northern blot? Compare this with the information obtained from a Southern blot.

Question 39

Describe fluorescence in situ hybridization (FISH). When would you use this method, rather than Northern blotting?

Question 40

Design an experiment to quantify transcript levels for a particular mRNA using RNase protection assay (RPA). Show sample results.

Question 41

Explain the molecular basis of an RNase protection assay. How can this method be used to map the transcription start site for a gene?

Question 42

What kinds of information can be obtained from a Western blot? Diagram the key steps in the method.

Question 43

Explain the molecular basis for protein separation by isoelectric focusing (the first step of 2D-PAGE).

Question 44

You have used a polyclonal primary antibody made in rabbit to detect your protein of interest on Western blot. Give an example of an appropriate secondary antibody to use for detection. Explain your choice.

Question 45

Compare and contrast the steps in production of polyclonal and monoclonal antibodies.

Question 46

Diagram the key steps in a "sandwich" ELISA. What information can be obtained from this type of assay?

Question 47

Compare and contrast the electrophoretic mobility shift assay (EMSA) and DNase I footprinting methods for assaying specific DNA-protein interactions. What information does DNase footprinting provide that EMSA does not?

Question 48

You want to confirm the interaction of a particular protein with a specific sequence of DNA within the context of a living cell. What technique would you use and why?

Question 49

You want to confirm the interaction of a particular protein with another protein at a precise location within a cell. What technique would you use and why?

Question 50

Diagram the key steps in a GST pull-down assay. Is the GST-tagged protein the "bait" or the "prey"?

Question 51

In a yeast two-hybrid assay that uses lacZ as a reporter gene, what does the production of a blue product signify?

Question 52

Compare and contrast the type of information that can be obtained from X-ray crystallography with nuclear magnetic resonance (NMR) spectroscopy.

Question 53

Compare and contrast the type of information that can be obtained from cryoelectron microscopy with atomic force microscopy.

Question 54

You have purified a protein. When you subject it to SDS-PAGE, two bands are seen. Provide a possible explanation and describe how you could test your hypothesis.

Question 55

Consider a Northern blot:
(a) Which strand of a DNA probe will hybridize to mRNA on the blot: the anti-sense strand, the sense strand, or both strands?
(b) On the diagram of the DNA-RNA hybrid shown below, which bonds form when the DNA probe hybridizes to the mRNA?

Question 56

Consider a cotransfection assay with expression vectors for two putative transcription factors that you suspect work together to activate transcription, and with a regulatory region of interest attached to a CAT reporter gene. You also test two deletion () mutants of the regulatory region.
(a) How could you confirm that the transcription factors are transcribed and translated in the transfected cells?
(b) Interpret the results of the CAT assay below. The plus and minus symbols indicate that presence or absence of expression vectors for the listed components in the transfection assay.
(c) How would you demonstrate protein-protein interaction between the two transcription factors in vivo and in vitro? Show sample positive results.
(d) Describe protocols to further characterize direct binding of the proteins to DNA in vitro and in vivo. Show sample results.

Question 57

You have transfected siRNA against transcription factor A from Q. 59 into cells that normally express the transcription factor.
(a) What would be the effect on CAT reporter gene expression?
(b) How could you determine whether RNAi acts at the level of mRNA or translation in this case?