The virtual laboratory: Mutations in an exon
copyright © 1982 - 2006 David A Bender
Virtual Laboratory main menu
Click here to run the program
The program will run in a separate window, and at any time you can minimise the program window to check the theory from this page.
Sequencing the exon
Screening the mutants
Spuriosum hypotheticum is a pathogenic yeast that is resistant to most currently available anti-fungal agents.
It has an unusual coenzyme, coenzyme X, in one of its main energy-yielding pathways, and the enzyme responsible for the synthesis of this coenzyme, coenzyme X synthetase, has been much investigated as a possible target for a therapeutic agent.
The gene for coenzyme X synthetase has been cloned, and one exon, consisting of 24 base pairs, codes for a peptide sequence that seems to be involved in substrate binding, and hence catalytic activity. This exon also seems to be a mutation "hot spot", in that exposure of the organism to uv light results in a number of mutants in this exon, some of which result in partial or complete loss of enzyme activity.
Your task is to determine the amino acid sequence coded for by this exon in the wild-type organism, then to investigate the changes in the peptide in a number of mutant strains produced by uv irradiation.
Sequencing the exon
When you start the program and click on the button to sequence the exon, you will see the base sequence of the exon, as it appears in the mRNA. You have to decode the sequence to identify the amino acids in the peptide.
The table shows the genetic code, as in mRNA. Click here to download a printable version.
When you have identified all the amino acids in the peptide coded for by this exon, you have to calculate the charge on the peptide at around pH 7 - this will allow you to determine whether or not the variants of this peptide in the mutants that you will work with later could be separated by electrophoresis.
The diagram below shows the structures of the amino acids. Click here to download a printable version
Screening the mutants
You can prepare a variety of mutant organisms by exposing a culture to uv irradiation, then plating a dilute suspension onto an agar gel containing medium supplemented with coenzyme X. This will allow all the viable organisms to grow.
These colonies can be blotted onto a fresh agar gel, containing minimal culture medium - i.e. not supplemented with coenzyme X. Comparison of the two plates will allow you to see:
which colonies have active coenzyme X synthetase - they grow as well as on the supplemented medium
which colonies have impaired activity of coenzyme X synthetase - they form smaller colonies than on the supplemented medium
which colonies have no activity of coenzyme X synthetase - they do not grow at all (and are shown in the example below by open circles to mark where there are viable colonies on the plate containing supplemented medium)
You select a colony to study by clicking on it on the left-hand (supplemented medium) plate; this allows you to investigate the change in the exon of interest in each mutant strain of Spuriosum hypotheticum.
By comparing the amino acid sequence in the wild-type and the mutant strains, and the effect this has on viability on minimal medium, you can begin to draw conclusions about the active site of the enzyme.