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(Continued from page 1)
Isolation
of lac repressor.
Gilbert and Muller-Hill, Proc. Natl. Acad.
Sci. 56:1891,
1966. What was the primary obstacle to isolating the repressor
and how was it overcome? Try to figure out the selection for the
it mutants; don't worry about the details of the plot
shown in Figure 4. What other properties of repressor were yet to
be determined?
Isolation of catabolite repression mutants. Schwartz and
Beckwith, in The Lactose Operon,
p. 417. Be able to define catabolite repression. Figure out
the selection and how adenyl cyclase mutants were distinguished
from CAP mutants. Were the authors correct in saying that genetic
tests cannot be used to determine whether CAP acts negatively or
positively?
An in vitro system for studying lac
expression. Zubay et al., Proc. Natl.
Acad. Sci. 66:104,
1970. Concentrate on Table 1. Understand the components required
for DNA-dependent in vitro protein synthesis and the general
way in which such systems are used for isolating proteins. What
are the limitations to this in vitro complementation approach?
What would the results have been if CAP were a negative control
protein, i.e., if the pleiotrophic, carbohydrate-negative mutations
defining CAP were analogous to lac super-repressor mutations?
Genetic evidence for the bipartite
structure of the lac
promoter. Beckwith et
al., J.
Mol. Biol. 69:155,
1972. Be able to diagram each cross in Tables 2 and 3. What
do the two domains of the promoter do? What mutants are required
to confirm the model presented and what would their respective phenotypes
be?
Initial models of the mechanisms
of action of lac
repressor and CAP. Summarized
in Reznikoff and Abelson, in The
Operon,
p. 221-243. Dr. Wolf will lead the discussion of Figs. 3-6.
Basic techniques of molecular
genetics. A set of handouts
will be used by Dr. Wolf to describe the Maxam-Gilbert method of
DNA sequencing and its application to studying studying protein-DNA
interactions by chemical protection and interference assays. Other important techniques for studying protein-DNA interactions will also be described, e.g., DNase I footprinting and gel retardation assays.
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Protein-DNA interactions in the lac operon.
Repressor-operator interaction. Ogata and Gilbert, Proc. Natl. Acad. Sci. 74:4973, 1977. Be able to explain the method used to probe the protein-DNA interaction. How was the DNA radiolabeled on one end of one strand?
(Continued on page 3)
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