Download Fall06MicrobGenetExamI

Name_______________________________
Student ID#__________________________
MicrobialGenetics,
Bacterial
Genetics,BIO
4443/6443
BI
410/510
Fall 2006,
Semester
Exam
2001
I
Exam I
1.) Draw the structures thymine and uracil. (5pts)
2.) What happens when cytosine deaminates in the DNA? Why is this a problem for the cell?
(3pts)
3. ) An unknown replication enzyme is added to the following substrate and generates the
following product. What is the enzyme called? (5pts)
UNKNOWN
ENZYME
4.) By itself, PolIII, encoded by dnaE, incorporates the wrong base approximately once in every
105 bases. The measured rate that a misincorporated base results in a mutation is actually closer
to once in every 1010 bases. Describe two general mechanisms or activities that operate to
increase the fidelity of replication. (8pts)
Name_____________________
page2
5.) tus and ter are involved in what cellular process? How do they function? (4pts)
6.) Determine whether the following are examples of transitions or transversions? (4pts)
a GC base pair changes to a CG base pair __________________
a AT base pair changes to a TA base pair __________________
a GC base pair changes to a TA base pair __________________
a AT base pair changes to a CG base pair __________________
7.) You have found a gene, yebC that you know is turned on when the cells are exposed to UV
light. You wish to know more about the yebC gene and its function. A colleague of yours has
three strains that each have single base mutations in the third codon of the yebC gene. One strain
contains a missense mutantion, one strain contains a nonsense mutation, and one strain contains a
frameshift mutation. The colleague asks which strains you would like to use in your studies.
Which mutant is the LEAST likely to give you a complete lack of function (i.e. knockout
YebC function)? Why? (4 pts)
8.) You have isolated two mutants that do not contain any ß-galactosidase (the mutants do not
grow on plates that have only lactose as a carbon source). They are interesting because when
you sequence the gene for ß-galactosidase in these mutants, neither mutant contains a mutation
in the coding region. You decide to sequence upstream of the start codon and find that mutant
#1 has a deletion within the TATA box. Mutant #2 contains a point mutation in the Shine
Delgarno sequence.
How can mutations in the TATA box or Shine Delgarno sequence result in a failure to
express any ß-galactosidase enzyme? (6pts)
Name_____________________
page3
In their classic experiment Matt Meselson and Frank Stahl demonstrated that DNA replicated
semi-conservatively by density labeling the DNA and separating it in CsCl gradients.
A.) For one control, they grew one culture of E.coli in media containing heavy isotopes
of nitrogen (14N) and carbon (13C) to label the DNA during growth.
B.) For another control, they grew another culture of E.coli in normal media.
C.) For their experimental analysis, they grew a third culture in the media containing
heavy isotopes. Then, after several generations of growth in this media, they transferred the
bacteria into normal media and prepared samples at various times ( I. immediately before
transfer, II. after one doubling time in the normal media, and III. after two doubling times in the
normal media).
They lysed the cells, isolated the DNA, and centrifuged each sample to equilibrium in
neutral CsCl gradients. Shown below is where their control DNA samples A and B were found
in the gradients.
For each case, draw in where the DNA band(s) should appear for their three time points:
9.) If replication were to occur by a semiconservative mechanism: (4pts)
A.
"Heavy"
media
B
Normal
media
CI
Immediately
before
transfer
CII
After one
doubling
time
CIII
After two
doubling
times
10.) If replication were to occur by a conservatively mechanism: (4pts)
A.
"Heavy"
media
B
Normal
media
CI
Immediately
before
transfer
CII
After one
doubling
time
CIII
After two
doubling
times
Name_____________________
page4
11.) If replication were to occur by a distributive mechanism (4pts)
A.
"Heavy"
media
B
Normal
media
CI
Immediately
before
transfer
CII
After one
doubling
time
CIII
After two
doubling
times
Recently, a group of scientists found that a rare genetic disease causing chronic fatigue in
humans is caused by certain mutations in the tryptophan biosynthetic pathway. You decide to try
and develop a model system in E.coli to study this disease by isolating mutants that are unable to
make tryptophan.
12.) Briefly describe how you would isolate trp- mutants. (6pts)
13.) Does this screen involve a positive selection or a negative selection? (2pts)
14.) In a screen of 10 million cells, you isolate 7 mutants. To determine how many different
genes you have isolated mutations from, you decide to do a complementation analysis. You
construct F’ strains containing each mutant before mating each combination and asking if the
two mutations complement each other. You results are shown below.
How many different genes are represented by your seven mutants? (5pts)
Name_____________________
page5
15.) One mutant, trpF, seems to be less motile when moving towards a food source. Thinking
that this may shed some light on the fatigue phenomenon, you decide to learn more about how
this mutation works by isolating suppressor mutations of trpF.
Briefly describe how you could isolate trp+ suppressor mutants from trpF. (6pts)
16.) Does this screen involve a positive selection or a negative selection? (2pts)
17.) You screen 10 million more cells but fail to isolate any suppressors. Why might the
reversion rate of trpF to trp+ be so much lower than the mutation rate for trp+ to trp- ? (4pts)
18.) What are two strategies you could use to increase the likelihood of isolating a trp+
suppressor from your trpF culture in your next screen? (4pts)
Name_____________________
page6
Luria and Delbrück were trying to come up with an experiment to differentiate between the
random-mutation hypothesis and the directed-change hypothesis in bacteria. In the experiment
they came up with, they utilized the generation of resistance in E.coli to infection by phage T1 as
their assay. The following represents how one of their experiments may have been performed.
In the first part of their experiment, they innoculated a single 20 ml culture with bacteria
that were sensitive to phage T1. They then let the culture incubate overnight. They next day
they plated 1 ml aliquots of the culture on thirty plates containing the T1 phage and then counted
how many resistant colonies were able to grow on each plate.
Their results are shown below:
plate#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
T1
resistant 13 14 12 14 12 13 15 12 13 13 14 12 14 12 13 15 12 13 12 11
phage
In the second part of this experiment, they inoculated 20 individual 1ml cultures with
with bacteria that were sensitive to phage T1. They then let the culture incubate overnight. They
next day they plated each 1 ml culture on a plate containing the T1 phage and then counted how
many resistant colonies were able to grow on each plate.
Their results are shown below:
plate#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
T1
resistant 1 0 2 0 8 0 15 0 13 0 156 43 55 6 113 15 3 34 2 18
phage
19.) Based upon the above results, they concluded that spontaneous mutations must occur in the
culture. Why? (4pts)
20.) Assuming that the random mutations are the only source of mutations in this population,
calculate the mutation rate from the above experiment if the cultures contained 109 cells/ ml.
Remember that the mutation rate, a
= m/N and use the Poisson expression where the
probability of having i mutations per culture is represented by Pi = ( mi e-m )/ i!. Show
your work. (10pts)
21.) Hand in your “Recombineering”primer sequences that could be used to and replace the
recF gene with a gene encoding kanamycin resistence on the E. coli chromosome. (10pts)