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MCB 421
Homework 10
Due Thur Nov 24
Fall 2011
1. CTnDOT is a conjugative transposon originally found in Bacteroides. Work on the element produced
the DNA sequence (attDOT) of the region involved in site-specific recombination of the element. The
DNA sequence of the attDOT site is on the last page of the homework. Analysis of the sequence attDOT
and bacterial (attB) sequences showed that the recombination occurs between attDOT and attB by
staggered cleavages seven base apart on each att site. The sites of cleavage in attDOT are shown between
the D and D’ sites in the sequence.
In vitro experiments indicated that the IntDOT integrase, which catalyzes the reaction, binds to two classes
of sites in attDOT. One class, called core type sequences, are represented by the D and D’ sequences on
the sequence. These sites are thought to be sites where IntDOT binds to cleave the DNA during strand
exchange. A second class of sites, called arm sites, are labeled as R1, R2, R2’, L1 and L2 in boxes in the
sequence. These sites are thought to be bound by IntDOT to form nucleoprotein complexes required for
either integrative or excisive recombination. If that hypothesis is correct, mutations of the sites should
affect either the integration or excision reaction because the protein won’t recognize the mutant site. For
example, mutation of L1 might abolish integration but have no effect on excision etc.
In order to test this hypothesis, you decide to mutate the L1 site and test the mutant attDOT site in
integration and excision assays. Assume that you have attDOT cloned on a pUC-based vector and that you
have assays for both integration and excision of any mutant you make.
a. You decide to use the Quickchange method to change all six basepairs in the box in the L1 site using
oligonucleotide mutagenesis. Design the sequences of the oligonucleotides showing the changes in the L1
site and the six bases on the 3’ and 5’ sides of the box. Make sure to clearly label the 5’ and 3’ ends of the
oligos. In a real experiment how long would the oligos be?
b. Why is the template grown in a dam+ cell?
c. Why is the DNA treated with DpnI after the DNA synthesis step?
2. You have isolated a novel operon, the vir operon, that contains two genes required for virulence in
Salmonella. You have constructed operon fusions with the galK gene (encoding galactose kinase) and gene
fusions with the lacZ gene (encoding ßgalactosidase) for each of the two genes, virF and virG. The
expression of each fusion was assayed with or without exposure to superoxide (superoxide is an oxygen
radical that pathogens can encounter in hosts). The results are shown in the following table.
Gene
galK operon fusion
Galactose kinase activity
-superoxide
+superoxide
lacZ gene fusion
B-galactosidase activity
-superoxide
+superoxide
virF
40
40
40
400
virG
5
45
50
450
a. What do the results indicate about the regulation of the virF and virG genes? Briefly explain your
answer.
3. You are studying an interesting operon, the sitAB operon, that contains two genes required for virulence
in Salmonella. You have constructed operon fusions with the galK gene (encoding galactose kinase) and
gene fusions with the lacZ gene (encoding ß-galactosidase) for each of the two genes, sitA and sitB. The
expression of each fusion was assayed with or without exposure to superoxide (superoxide is an oxygen
radical that pathogens can encounter in hosts). The results are shown in the following table.
What do the results indicate about the regulation of the sitA and sitB genes? Briefly explain your answer.
MCB 421
Homework 10
Due Thur Nov 24
Fall 2011
4. You have isolated a new gene, yssA, from Salmonella. After obtaining the DNA sequence of the gene
and performing a GenBank search you find that the gene has homology to known Type III topoisomerases
from a variety of bacteria. It has a tyrosine residue that is part of the active site of all known Type III
topoisomerases. In described topoisomerases, this residue is required for activity. Assume that you have a
method to isolate YssA protein in small quantities and that you have detected topoisomerase activity in
YssA isolates. Assume that you have a simple method to assign a positive or negative phenotype for
isolated YssA protein (i.e. you can say whether a given version of the YssA protein is functional or not).
a. Explain how you would use the Quickchange PCR method of site-directed mutagenesis to test whether
the conserved tyrosine residue is required for YssA function. Draw a diagram showing where each primer
would anneal on a plasmid containing yssA. Show the location of the mutagenic nucleotide on each primer
in your diagram.
b. What would you want to do with the Quickchange PCR reaction after PCR and before transformation?
Why?
c. If the relevant Tyr codon is UAU, suggest a one-nucleotide change you could make to change the amino
acid using the above protocol. What new amino acid would this result in? Do you think this change in
amino acid would definitively test the importance of the Tyr residue? Why or why not?
d. Describe a genetic experiment that could help you purify a large amount of the protein for biochemical
studies.
e. What is one possible problem with the experiment described in part d?
MCB 421
Homework 10
Due Thur Nov 24
Fall 2011