Home > Focus stories > Evolved enzymes> Q&A: Directed
evolution and cancer therapy
Directed evolution and cancer therapy: Question sheet
1.
What is the difference between selective breeding and directed evolution?
2.
In the first step of cancer prodrug therapy, the gene coding for the enzyme is
cut out of the bacterial cell’s genome. What type of enzyme is used to do
this?
3.
How do restriction enzymes recognise where to cut DNA?
4.
The viral vector DNA is cut, and the bacterial gene, which codes for the
enzyme, is spliced into the viral DNA. What types of enzymes are used for
these steps?
5.
Suppose the restriction enzymes Eco R1 and Bam H1 were used to cut either
end of the gene for the enzyme from the bacterial genome. Explain why these
same restriction enzymes are used to cut the viral DNA.
6.
How does DNA ligase join the two DNA fragments together?
7.
The virus now has a bacterial gene in its DNA. What is the term for DNA that
has DNA from another species inserted into it?
8.
What is the role of the viral vector in this form of gene therapy?
9.
Directed evolution induces mutations in the bacterial gene coding for the
enzyme that activates the prodrug. Each mutation affects the gene in a
different way, for example, by deleting or inserting nucleotide bases. Briefly
explain how such mutations affect the resulting enzyme protein coded for by
the mutated gene.
10.
The enzyme activates the prodrug and produces a toxic metabolite that
crosslinks the DNA strands in the cancer cell. What effect would crosslinking
the DNA have on the cell?
11.
The enzyme activates the prodrug to produce a toxic metabolite that
ultimately kills the cancer cell. Surrounding cells are killed by the bystander
effect. Explain what is meant by ‘bystander effect’.
12.
What safeguards are there in place to reduce the impact of virus-directed
gene therapy on the cancer patient’s healthy cells?
©2005-2009 The University of Waikato
www.biotechlearn.org.nz
Home > Focus stories > Evolved enzymes> Q&A: Directed
evolution and cancer therapy
Directed evolution and cancer therapy: Answer sheet
1.
In selective breeding, one selects for certain phenotypic traits and uses
breeding (sexual reproduction) between individuals to enhance or improve
these traits. In directed evolution, one selects at a molecular level for a gene
that encodes an enzyme. Mutations are introduced into the gene to change its
function. The gene variants that code for enhanced enzyme function are then
selected.
2.
Restriction enzymes (or endonucleases).
3.
Each restriction enzyme has a specific base sequence recognition site, of four
to eight base pairs, where it cuts the DNA.
4.
Restriction enzymes and DNA ligases.
5.
The same restriction enzymes would be used to cut the bacterial DNA and the
viral DNA so that the two fragments of DNA would have matching sticky ends,
which can adhere together by base pairing. In this way, the bacterial DNA can
be inserted into the viral DNA.
6.
DNA ligase joins the DNA fragments together by forming phosphodiester
bonds between the sugar and phosphate molecules of the DNA backbone.
7.
Recombinant DNA.
8.
The viral vector seeks out cancer cells and inserts its DNA into the cancer
cells’ DNA.
9.
The mutations in the DNA produce different base sequences in the DNA of the
gene. These, in turn, affect the amino acids coded for by the gene. Therefore,
the resulting enzyme proteins will have different amino acid sequences. This
can affect the structure of the enzyme protein, the folding of the protein chain
and the physical structure of the active site, which, in turn, affect the
enzyme’s function.
10.
Crosslinking the DNA means that it cannot separate freely to replicate. If the
DNA cannot replicate, the cells cannot divide by mitosis, and no daughter
cells can form. Also, if DNA cannot separate, no mRNA can be transcribed.
Therefore, no enzymes or other proteins can be formed, and the cell cannot
function. Ultimately, the cell dies.
11.
The toxic metabolite produced in the cancer cell is able to move through the
cell membrane into surrounding cells. Here, the toxic metabolites act on the
DNA of these cells and prevent the DNA in these cells from functioning,
ultimately causing these cells’ death too. This is the ‘bystander effect’.
12.
The virus is disabled, so it can only infect cancer cells. The virus cannot
replicate in normal, healthy cells. A bacterial enzyme is used, which is not
©2005-2009 The University of Waikato
www.biotechlearn.org.nz
Home > Focus stories > Evolved enzymes> Q&A: Directed
evolution and cancer therapy
normally present in human cells. The prodrug itself is harmless, unless
activated. Both the virus and the prodrug must be present in the cancer cell
for the toxic metabolite to be produced.
©2005-2009 The University of Waikato
www.biotechlearn.org.nz