| M W F; 10:10 - 11:00 pm |
Molecular Biology |
Douglas W. Smith |
| York 2722 |
BIMM 100 |
5254 Muir Biology Building |
| Fall, 2000 |
x42620; dsmith@ucsd.edu |
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
What is a YAC? What is the meaning of each word in the acronym YAC?
What are YAC "arms"?
Why were YACs developed, ie why are they an important class of vectors?
What is a cloning vector or cloning vehicle?
What is meant by cloning?
What is the size range of DNA which can be cloned in a YAC? How does this compare with other types of cloning vehicles?
What is pulsed-field gel electrophoresis? How does it differ from normal slab gel electrophoresis? What is meant by "slab gel"? For what types of DNA is it advantageous to use pulsed-field gel electrophoresis? Why?
The authors state "many of the functional genetic units in higher organisms span enormous tracts of DNA." Why is this? Why is it not true in bacteria?
Why, in a mapping experiment, is it desirable to have fewer, larger clones rather than many, smaller clones? When do smaller clones become necessary?
What are the genetic features of pYAC2? What are the physical features? What is (are) the function(s) of each of these features?
How does cloning into a YAC differ from cloning into a plasmid vector?
What types of DNA were cloned in this paper? What experiments showed that these types of DNA had been cloned? How was presence of the YAC itself determined? From what DNA did the cloned chimeric DNA need to be distinguished?
What is a chimeric DNA molecule?
What is the usual host in a YAC cloning experiment? What other hosts could be used? Why is a host used in a cloning experiment?
What is a Southern gel? What is analyzed? How is it analyzed?
What is a Northern gel? What is analyzed? How is it analyzed?
What is P32 ? How is it analyzed? Why is it useful in biology? What other isotopes are useful in biology? Why is two weeks normally an upper limit on time used to detect a P32 signal? What would the upper time limit be for C14 ? for N14 ?
In a cloning experiment, why is ligase used at 15·C or lower? why is it used for 12-15 hrs? what does the ligase do?
Why are markers usually present in one or two lanes of a slab gel experiment? Why are they present in more than one lane? Why are they often present in the outer lanes used?
Why do some yeast strains transform more readily than others? What is meant by "transform"?
How was it shown that the cloned Yeast SmaI fragment in YY1 has a SmaI couterpart fragment in the yeast genome? Why was SmaI used in this comparison experiment?
What is indirect end-label-mapping? How was this used to obtain information regarding BamHI sites in the cloned insert? Why was a "partial" digest used?
What new features are present in the more recent vectors pYAC3, pYAC4, pYAC5?
Why is there any doubt that there exist metazoan origins of DNA replication?
How has yeast (S. cerevisiae) been used as a 'paradigm' for the eukaryotic initiation process?
What is a metazoan 'Initiation Zone'? What are its properties, i.e. what does it contain?
What 'multiple factors' are involved in origin establishment? How do they work?
Define a replication origin.
What do organisms do to increase their rate of DNA replication?
Why do eukaryotic chromosomes have multiple origins of DNA replication?
What is the Cell Cycle of a cell of an organism?
At what time during the Cell Cycle does DNA replication take place?
When during this time does initiation take place?
At what time during the development of a higher organism does initation appear to take place at random DNA sites? Why is this the case? (see Lodish, Fig. 10-5)
What are ORC proteins and what do they do?
What are MCM proteins and what do they do?
What are yeast cdc mutants?
What reactions do 'kinase' proteins catalyze? What kinase reactions are important in eukaryotic initiation?
What is SV40? What is the structure of the SV40 replication origin? What proteins are important in initiation from the SV40 rep origin? What protein is a cognate to the E. coli DnaA protein?
What is the structure of a typical yeast origin of DNA replication (ARS)? ... (see Lodish, Fig. 10-8)
What does ARS stand for?
What is a DUE and what does it stand for?
Are all ARS true origins of DNA replication? Why or why not?
What are the two classes of experiments that have been used to attempt to identify metazoan rep origins?
What is a metazoan?
What hypothesis does DePamphilis put forward to attempt to explain why the two classes of experiments yield results regarding metazoan rep origins that differ from each other?
What is an OBR and what does this mean?
Why is a rep origin region from S. pombe a reasonable paradigm for a metazoan rep origin?
What is an Initiation Zone?
What are the main parameters that appear to determine origin specificity? ... see Fig. 3
What are the three main roles that Nuclear Structure is thought to play in origin specificity?
What is the Origin Decision Point? ... see Fig. 4
What role does Chromatin Structure play in origin specificity determination?
How important is DNA sequence in origin specificity?
Give an example where DNA sequence plays little or no role.
Give an example where DNA sequence plays a very important role.
Are metazoan origins of DNA replication genetically inherited?
What function do DNA replication origin sequences play?
What two roles do transcription events play in determining origin specificity?
What type of methylation of DNA occurs in eukaryotes? is this different from prokaryotes such as E. coli?
What is a 'CpG island'? how are CpG islands related to DNA methylation in eukaryotes?
What evidence suggests methylation is important in some eukaryotic initiation events?
What evidence indicates methylation is not important in other eukaryotic initiation events?
What are the likely minimal DNA structural requirements of a metazoan replication origin?
Where is the OBR likely to be relative to the ORC binding site and the DUE?
What is photoreactivation and how was it discovered?
What is photolyase and what reaction does it catalyze?
What is excision repair?
What DNA damage is recognized by excision repair processes?
What is the basic enzymatic mechanism used in all excision repair processes?
How does this basic enzymatic mechanism differ between organisms?
What is the role of the "DNA template" in excision repair? What is this "template"?
Understand base excision repair.
What reaction is catalyzed by a DNA glycosylase? Name two DNA glycosylases?
What is an AP nuclease? What does AP mean? How does AP DNA arise?
Understand nucleotide excision repair.
What is a DNA "excinuclease"? How does it differ from a DNA endonuclease? DNA exonuclease?
Comment on the statement: "An Excinuclease is a single protein catalyzing a single reaction".
What specificity is shown toward DNA damage that can be recognized by an excinuclease? What is good about this "specificity"? What is bad about this "specificity"?
Understand what the UvrA, UvrB, and UvrC proteins do in E. coli.
Which of these proteins is an ATPase? What is an ATPase? Why is an ATPase needed in excision repair?
Which of these proteins are "nickases"?
How is Helicase II (the UvrD protein) involved in E. coli excision repair? What is a helicase?
What reaction of PolI is used in the UvrABC nucleotide excision repair?
Design and describe a Nucleotide Excision pathway that would use a SINGLE nick in the damaged DNA strand and would use TWO activities of PolI.
What are the major proteins in the human excinuclease?
Which of these are the cognates to the UvrA, UvrB, and UvrC proteins?
How are they related to human disease?
What are the important human diseases here?
What protein parts of the human Nucleotide Excision complex are also used in transcription?
What protein parts of the human Nucleotide Excision complex are also used in replication?
How does use of common proteins in these processes provide coordination of these processes?
Why is Pol-delta sometimes used in repair, but Pol-alpha is used very seldom?
What is transcription-coupled DNA repair?
Why is it advantageous to have a specific repair system for DNA containing expressed genes?
What is Cockayne's syndrome, and how is it related to transcription-coupled DNA repair?
What is the primary difference between transciption-coupled repair in prokaryotes and in eukaryotes?
What is eukaryotic transcription factor SII and what does it do?
How is this SII activity relevant to a mechanism for transcription-coupled repair in eukaryotes?
What is mismatch repair?
What is significantly different about the type of DNA "damage" repaired by mismatch repair compared to other types of DNA damage?
Understand what the MutH, MutL, and MutS proteins does in E. coli.
How is the "strand recognition problem" in mismatch repair solved in E. coli?
How is the "strand recognition problem" in mismatch repair solved in humans?
Is there a difference between the UvrD and MutU proteins in E. coli? Explain why.
In MutHLS mismatch repair, where does the initial nick take place?
ExoI degrades 3'->5' and is specific for ssDNA, whereas ExoVII degrades 5'->3' either ssDNA or dsDNA.
Why are BOTH enzymes needed for MutHLS mismatch repair?
HoloPolIII normally fills in the "gap" in mismatch repair, rather than PolI. Why do you think this is so?
What disease is mismatch repair correlated with?
What is the heat-shock response?
The htpR165 mutation is an amber mutation. What is an amber mutation?
This mutation is suppressed by a tRNA mutation. What is suppression? What type of suppression is this? How does this type of suppression work?
What evidence before this paper suggested that the HtpR protein was a sigma factor? What is a sigma factor? Given that this protein is a sigma factor, explain how it would be a control factor in regulation of the heat-shock response.
What was the experimental system used to examine effects of overexpression of the HtpR protein?
Why was the lambda PL promoter used? What is the lambda PL promoter? Why is this coupled with used of the lambda CI857 repressor? What is the lambda CI repressor? What is the phenotype of the CI857 mutant repressor?
Why does overexpression of the htpR gene result in overexpression heat-shock proteins? How was this experimentally shown to be true? What is an SDS gel? What is SDS? What does SDS do to proteins?
Why was plasmid pKWT5 constructed (see Fig. 2A)? What was it used for? Why does it contain the PHS promoter? What is the PHS promoter? Where is it found on the E. coli chromosome? Why do you think it is in this position of the E. coli chromosome?
What is a "runoff" transcription assay? How was this assay used with plasmid pKWT5? Why were two restriction sites used in this assay? How were they used?
Why were "crude extracts" used in this runoff transcription assay? What was the source of the curde extracts? What is a "crude extract"?
What is the purpose of the authors assaying the crude extract? What are they assaying for?
The crude extracts were prepared from treated E. coli cells. How were the E. coli cells treated? Why was this done?
What was the method for analyzing RNA products of the runoff assay? What controls were done? What are the Markers and why were they used?
What is the pBR322 ori transcript (p. 21, top right)?
What evidence indicated that "the activity that stimulated P transcription" fractionated with E. coli RNA polymerase? Why don't the authors simply call "the activity that stimulated P transcription" the HtpR protein? What is meant by "fractionated"?
How did the authors use this information that the activity fractionated with RNA polymerase?
What is the Coomassie blue stain assay? How was it used? Why is the transcription assay (Fig. 3B) used also?
"Reconstituted enzyme" was prepared from protein eluted from the Bio-Rex 70 column. How was this done? Have you any reservations about this procedure?
Why were experiments done using the dnaK P1 and P2 promoters? What were these experiments and what did they show?
Minicells were used to demonstrate that the sigma-32 protein was the htpR gene product. Why do the authors worry about this? Wasn't this shown already, in Figs. 1-4?
What are minicells? Why are they used? How are they used? In Fig 5, what does the S35 assay? what does the H3 assay? What are S35 and H3 ? How did the authors assay S35 itself? H3 itself?
What is HtpR'? How does it differ from HtpR? Why was it used?
What is 2-dimensional gel electrophoresis? Why was it used? What results did it show that were not shown on the 1-D gels?
How was the identity of sigma-32 protein finally made with HtpR protein? How convincing are these data?
What is the role of the sigma-32 protein is regulating the heat-shock response, both turn-on of the response and turn-off of the response? What two possible mechanisms are considered by the authors?
How can sigma factors be used to regulate gene expression in time, eg during sporulation in B. subtilis or during a viral growth cycle?
What is the MT gene and what is its function? What two types of molecules induce activation of the MT gene? What are the major results of this paper?
How does hMT-II A differ from MT-II A ? How does hMTK differ from hMT-II A ?
Activation of the MT gene results in increased levels of MT gene mRNA, and hence activation occurs at the transcription level. How would you show experimentally that activation results in increased levels of MT gene mRNA?
What methodology did the authors use to map the regulatory DNA elements? What properties of these DNA elements did they determine? What DNA elements did they find and how many copies of each? How do these compare with those shown in Lewin, Fig. 30.1?
Why did the authors use the hMTK fusion gene to generate deletion mutants? Why did they generate deletion mutants? How did they generate the 5' deletion mutants
EXPLANATORY NOTE: The use of the TK gene on the plasmid and growth of tk - fibroblasts in HAT medium is to select for the plasmid upon transformation of the fibroblasts. The plasmids used are pUC8 derivatives, and hence have no origins for replication in eukaryotic cells; the HAT medium provides a strong selection for the tk + gene on these derivative plasmids and they are recombined into one of the chromosomes in the fibroblast cells.
In Table 1, what effect did addition of dexamethasone to the growth medium have on transformation with plasmids containing the complete hMTK fusion gene? with each of the deletion plasmids? What conclusions could be drawn from this experiment? What happened to the transformation rate when all but 90 bp or less were removed from the 5' regulatory region? What upstream site had now been deleted?
How do the Northern gel analyses of Fig 1b confirm the results shown in Table 1? What is a Northern gel?
How does the S1 nuclease mapping experiment of Fig. 1c show that the same transcriptional start site is used by all derivative plasmids? How do these results also confirm the results shown in Table 1 and in Fig. 1b?
Why were 3' deletion derivatives of the MT regulatory region constructed (Fig. 2)? Why was it necessary to use a "hybrid" promoter region? What do the Northern analyses of these 3' derivative plasmids show (Fig. 2b)?
How do the analyses of the 3' deletion derivatives regarding Cd++ activation differ from the analyses of the 5' deletion derivatives? How was this discrepancy resolved?
Why was the binding of glucocorticoid receptor to the 5' deletion derivative plasmids measured (Fig. 4)? What then is the mechanism of activation by a steroid hormone at the Glucocorticoid Response Elements (GREs)?
What three methods (Fig. 4b, Fig. 5a, Fig. 5b) were used to show that the GRE is also responsible for receptor binding to DNA?
What conclusions do the authors draw regarding a eukaryotic promoter vs a prokaryotic promoter?
What do you think of their final thoughts regarding how Response Elements and Transcription Factors activate transcription?
These questions are not yet done ...
| BIMM100 | Syllabus
| Sections / Off Hrs | Grading
Policy | DNASYSTEM
|
| Lectures | Journal
Articles | Study Qs | Lab
Techniques | Exams |
If you have problems or comments, send email to Doug Smith