Name_________________________________________
Section_________
7.012 Problem Set 4
Question 1
You are studying the synthesis of the amino acid tryptophan in bacteria. The enzymes TrpA,
TrpB, TrpC, TrpD, TrpE and AroH are all required for tryptophan synthesis. In the presence
of tryptophan, wild-type bacteria do not synthesize any of these enzymes; however, in the
absence of tryptophan, all of these enzymes are synthesized at high levels.
a) Theoretically speaking, if the synthesis of the above enzymes is negatively regulated
i) what change to the repressor protein would cause the enzymes to be synthesized
even in the presence of tryptophan?
ii) what change in the operator sequence would cause the enzymes to be synthesized
even in the presence of tryptophan?
iii) what change in the repressor protein would cause the inhibition of enzyme
synthesis, even in the absence of tryptophan?
b) If the synthesis of the above enzymes is positively regulated,
i) what change in the activator protein would cause the enzymes to be synthesized
even in the presence of tryptophan?
ii) what change in the activator protein would prevent synthesis of the enzymes, even
in the absence of tryptophan?
iii) what change in the operator sequence would prevent synthesis of the enzymes,
even in the absence of tryptophan?
Question 1, continued
c) By mutational analysis you identify two regions of DNA that are important in the regulation
of tryptophan synthesis. The first of these regions, called trpR, is a gene that encodes a DNAbinding protein. The second region is a DNA sequence to which the trpR gene product binds,
called trpO. Analysis of three bacterial strains with different genotypes at the trpR and trpO
loci yields the following results:
i) Is the control of tryptophan synthesis likely an example of positive or negative
regulation?
ii) Is the protein made from the trpR gene an activator or a repressor?
d) Experiments to monitor the presence or absence of enzyme made from the trpC, trpD, trpE
and aroH genes in some isolated mutants yields the following results:
i) List all mutants that have mutations which affect production of any functional TrpC?
ii) List all mutants that have mutations which affect production of any functional TrpD?
iii) List all mutants that have mutations which affect production of any functional AroH?
Question 1, continued
e) Is the above data consistent with the theory that any of these genes are in the same operon
(and therefore under the control of a single promoter)? Why? Which genes would be
contained in this operon?
f) How could you explain mutant 5 in terms of regulatory mutations?
Question 2
While working with mice as a UROP, you discover a very important catabolic enzyme. You
make mutations in the gene encoding this enzyme such that the mutant enzyme is always
active. Mice carrying this mutant gene show rapid and permanent weight loss with no other
side effects. YOU HAVE DISCOVERED THE MAGIC BULLET THAT MILLIONS OF PEOPLE
WORLDWIDE HAVE BEEN SEEKING FOR GENERATIONS! But… you need the human
version of this gene.
To begin, you choose to make a human genomic DNA library.
a) In the space below, briefly outline how you would make a genomic DNA library in bacteria.
Include the terms: genomic DNA, restriction enzyme, plasmid, ligation, transformation, Petri
plates, probe, and hybridize.
Question 2, continued
You are successful in finding the human homolog. You have pinpointed the colony that
contains a plasmid, called plib1, with the human homolog. You isolate the plasmid, clone the
gene and designate it the svelte gene. Ideally, you want to express lots of human enzyme so
that you can do a complete biochemical study of the protein. Therefore, you need to move
the svelte gene from the plib1 plasmid into an expression vector. You have a great expression
vector, p7.01MIT, for use in E. coli. A diagram of the expression vector p701MIT and the plib1
are shown below with their unique restriction enzyme sites.
b) You want to insert a DNA fragment containing both the svelte gene and the kanamycin
resistance gene (kanR) into the p701MIT expression vector.
i) What enzyme(s) would you use to cut plib1 to obtain a single fragment containing
both the svelte gene and the kanamycin resistance gene (kanR)? What size fragments
would you get?
ii) What enzyme(s) would you use to cut p701MIT? What size fragments would you
get?
c) After ligation, you transform your new vector p701MIT containing both the svelte gene and
the kanR gene into an E. coli strain.
i) Prior to transformation, this strain of E. coli should be... (circle all that apply.)
ampicillin resistant.
ampicillin sensitive.
kanamycin resistant.
kanamycin sensitive.
ii) How would you detect which cells have the vector containing the svelte gene?
iii) What is the advantage of including the kanR gene on the fragment that you cloned
into p701MIT?
Question 3
a) You isolate cells that behave as expected. You isolate the vector that these cells carry. To
confirm that this vector is p701MIT containing the svelte gene, you perform a series of
restriction digests. On the gel below, mark places where you expect to find bands. Label each
band with the number of bases that the fragment would contain.
Now that you have the human version of the svelte gene cloned and you can express the
protein, Science Magazine and biotech companies are knocking at your door! You could make
millions of dollars before graduating from MIT! However, a skeptical, bitter graduate student
tells you that it is always a good idea to sequence newly cloned genes to make sure that you
have what you think you have. You decide to sequence the svelte gene.
b) For the following single-stranded piece of DNA, give the sequence of a 10 base primer that
you could use to sequence this piece. Label the 5’ and 3’ ends of the primer. This primer
should hybridize to the first 10 bases on one end and be elongated in the sequencing reaction.
primer:
c) What is the sequence of the newly synthesized DNA? Include and label the primer, and
label the 5' and 3' ends.
Question 4
You are a genetic counselor at a major Boston hospital. A couple (shown in the pedigree
below as 1 and 2) comes to you and wants to know what the probability is that the child they
are expecting will have big ears.
Recently, the gene that results in big ears was cloned and sequenced. The region flanking the
ear size gene is shown below.
a) You want to amplify this gene from each parent using PCR. Give the sequence of two
primers for this PCR reaction. Label 5’ and 3’ ends.
primer A:
primer B:
b) Below is a schematic of the same region. On the diagram below draw where each primer
would bind.
c) In a schematic like that in b) draw the DNA strands that would be present after 2 rounds of
PCR. Include the primers where appropriate on each strand.
Question 4, continued
A restriction map of this region is shown below:
The PCR-amplified DNA from each family member was digested with Hind III, Nde I, and
Bgl II. The digested DNA was separated by gel electrophoresis and the results are shown
below.
d) Is the big-eared phenotype dominant or recessive over the normal phenotype? How did
you determine this?
e) Given this family pedigree and gel, what are the chances that individuals 1 and 2 will have a
child with big ears? Please explain.