Download Bordoli Discussion Questions

Biol 400
Crowe
Bordoli Discussion Questions
(Nuc Acids Res. 29: 589 (2001))
Writing Assignment due Monday October 10 (10 pts):
I. Read the Bordoli et al. paper (available for downloading from course web page under readings:
PlantBordoli.pdf).
II. In your own words, provide written answers to the questions in parts A, B and C below (1-2 pages
with 1.5 spacing)
III. Use the figure-specific questions on p. 2 of the assignment to help guide you through analyzing
each figure (do not include answers to these questions in the written assignment). We will be
discussing the figures in-depth in class on October 10. See the Table on the next page for the figure
you should focus on for the in-class discussion.
A. Read the Introduction section:
1. How is histone acetylation thought to activate transcription?
2. Are acetylated histones always associated with gene activation?
3. What is known about the role of p300/CBP in flies and mice?
4. What is the hypothesis being addressed by this study?
B. Read the Results section and in your own words write a QMOC (described below)
for each of the five figures in the paper (ignore Table 1):
Question (Q): One sentence that summarizes the question being asked in this figure (why was this
experiment important to include in this study?)
Method (M): One sentence that summarizes the method or approach the authors used to answer the
question (e.g. immunoprecipitation to look for protein/protein interactions)
Observations (O): One sentence that summarizes the data shown in the figure
Conclusion (C): One sentence that summarizes the major conclusion of the figure (answering Q)
C. Read the Discussion and answer the following questions:
1) Do you agree with the statement that PCAT2 is a homolog of p300/CBP? Why or why not?
2) Why do you think plants evolved more p300-like proteins than animals?
Table A - Figure Assignments For
In Class Discussion
Group
Assigned Figure
AA Green
Fig. 1
AA Blue
Fig. 1
AA Red
Fig. 2
AA Yellow
Fig. 2
AB Green
Fig. 3
AB Blue
Fig. 3
AB Red
Fig. 4
AB Yellow
Fig. 4
AC Green
Fig. 5
AC Blue
Fig. 5
AC Red
Discussion section
AC Yellow
Discussion section
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Class presentation
Each figure was assigned to 2 groups. During class, one group will be selected to give a 5 minute
presentation of the figure. The 2nd group will provide any additional analysis of the figure
Your presentation should include
1) QMOC for your figure
2) Relevance of your figure to the paper (why was this figure included in the paper?)
3) YOUR analysis of the figure (including the 2 points described below)
When analyzing a figure in a scientific paper ask yourself the following 2 questions:
1) What controls were used in the figure and what was their purpose?
2) Do I agree with the author’s conclusions? Why or why not (were proper controls performed? Was an
appropriate method used to answer the question? Was the experiment repeated? Was sufficient sample
size used? Was a statistical analysis performed to increase confidence in any observed differences?)
Use the questions below to guide your analysis of your assigned figure. Answers to these questions are
NOT to be included in the writing assignment.
Fig. 1
1) How were p300/CBP related Arabidopsis proteins identified? (see Methods)
2) What are the structural differences between human p300 and Arabidopsis homologs?
3) What do asterisks represent?
4) What is the importance of the different domains i.e. what are they known to do in p300/CBP (see discussion)
5) What is interesting about the domain structure of Arabidopsis versus human p300/CBP (see discussion)
Fig. 2A
1) What is being detected on this blot?
2) What is 25S and what is it a control for?
3) Why is it important that the bands are at least 5kb in size?
4) What are possible reasons for more than one band being detected in a single lane?
5) What portion of PCAT2 would you use as a probe for this blot? Why?
Fig. 2B
1) What is being detected by ethidium bromide in this blot?
2) What does each band represent?
2) What is GapC a control for?
3) What is in the “control” lane and what is its purpose?
4) Why do you think authors use a northern for Fig. 2A and RT-PCR for Fig. 2B?
Fig. 3A
Note: SDS-PAGE is a denaturing polyacrylamide gel used to separate proteins based on their size.
1) How are the proteins made that are being added to this assay?
2) What differences might exist between proteins made in this way and proteins isolated from a plant cell?
3) What is Coomassie dye detecting?
4) Why is it important that histones are detected in each lane in 3A?
5) Can you figure out which bands represent the GST fusion proteins? Do you think the same amount of GST
fusion protein is present in each lane of 3A?
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Fig. 3B
1) What proteins and/or other components are present in this assay?
1) Is this an in vitro or an in vivo assay?
2) What is the purpose of the GST alone control?
3) What is being detected by autoradiography in 3B?
4) Why are histones detectable in some lanes of the gel pictured in 3B and not in others?
5) What are some possible reasons for seeing no activity of PCAT1 in 3B? (Hint: read discussion!)
Fig. 4A
1) How were amino acids selected that would be mutated?
2) What is the meaning of the nomenclature “ML” or “WY” for the mutants?
3) What is being detected on the blot? (i.e. why are histones visible in some lanes and not in others?)
4) Why show GST-AT expression levels?
5) What are some alternative explanations for seeing no activity for a mutated protein?
6) Is data consistent with Fig. 3B?
Fig. 4B
1) What cells were used for this experiment?
2) How many different plasmids are transfected into each cell and what is the purpose of each?
3) How is transcription activity being measured in this assay?
4) What is the purpose of the reporter-alone control?
5) Why would fusion of a CBP histone acetyl transferase domain (AT) to the DNA binding domain of
the transcription factor Gal4 activate transcription of a Gal4-dependent promoter?
6) What additional information does Part B give you that you didn’t get in Part A?
Fig. 5
1) What is E1A and why was it used in this figure?
2) What is being illustrated in the diagram?
3) Why were dl1107 and dl 1101 chosen for this study (what is important about the regions that are
deleted in these constructs? Do they bind other proteins?
4) Is this an in vitro or an in vivo assay?
5) What protein is being detected on the blot? Why do you only see this protein even though other
proteins are present in the assay? i.e. what does the presence of a protein on the blot indicate?
Discussion
1) What do you think the most important finding was in this paper?
2) What conclusions can you make about the function of Arabidopsis p300/CBP in plants from the data
in this paper?
3) What additional experiments would you want to see performed to increase our understanding of the
function of Arabidopsis p300/CBP?
4) What remaining questions do you have about Arabidopsis p300/CBP?