Download Mutation and Recombination

Altering Genes: Mutation and Recombination
Natural mutation is a very rare event and can only be studied in organisms with very high
division rates (such as bacteria). A spontaneous mutation may be detected by using
selective media. For example, the antibiotic penicillin, could be added to the culture
media. If some of the aseptically transferred bacteria grow into a colony you can be sure
that those cells underwent a natural mutation event that caused them to become resistant
to the penicillin.
2. Induced - in this case the mutation rate is greatly increased since it is caused/induced
by any number of different types of mutagenic agents / mutagens.
Examples of mutagenic agents include different types of ionizing radiation (x-rays, ultraviolet, etc.) and various chemicals.
We will be attempting to induce a mutation in the lab today using UV light (Caution - the
UV can also mutate your own DNA. Do not expose your skin or eyes to direct UV
radiation!). UV radiation causes dimerization of adjacent thymine dimers in the DNA. It
also causes the hydration of cytosines. As a result, DNA polymerase that would copy the
DNA strand during DNA replication cannot “read” either the thymine bound in a dimer
or the hydrated cytosine. DNA may fall off the strand and terminate replication or may
use nucleotides with non-matching nitrogenous bases. In the latter case, the substitution
of one base for another will lead to a mutation. If a purine base gets substituted for
another purine base (or a pyrimidine for another pyrimidine), we talk about a transition. If
a purine base gets substituted for a pyrimidine (or a pyrimidine for a purine), the result
will be a transversion. If a base gets lost, we will have a frameshift mutation.
Part A: Ultraviolet-induced mutagenesis of Serratia marcescens.
Serratia marcescens is a bacterium that appears red at room temperature due to the ability
to make a red pigment called prodigiosin. For the production of this pigment, several
steps are necessary, all of which are catalyzed by enzymes. Each enzyme, of course is
made from the information found in a gene, a stretch of DNA that encodes the
information for making a protein. In this experiment, we will induce DNA mutations with
various amounts of UV light. If any gene that is involved in coding for an enzyme in the
pathway of prodigiosin synthesis gets mutated, the enzyme may not be functional. As a
result, the bacteria may not be able to make the red pigment (prodigiosin).
Part B: Bacterial Conjugation
For this experiment we will use two auxotrophic mutants of E.coli. Usually, bacteria are
able to synthesize all the amino acids they need to produce all their proteins. Auxotrophic
mutants are lacking this ability. For an auxotrophic mutant to survive, it will need to have
the amino acids it cannot make itself. E. coli A cannot make the amino acid methionine,
whereas E.coli B is unable to make the amino acid threonine. Neither mutant will be able
to survive on minimal agar (missing those amino acids). Both mutants will grow on
nutrient agar or BHI agar (brain-heart infusion) with contains many nutrients including
the amino acids mentioned above. Usually bacteria divide asexually through binary
© B. Woelker, 2008
fission. However, bacteria are also able to recombine. This method is called conjugation.
Some bacteria contain an extra-circular DNA called an F plasmid. F stands for fertility.
The strains containing the F-plasmid are also called “donor” or “F+”. On the plasmid is
the information to produce a pilus, which can create a cytoplasmic mating bridge to a
“recipient” or “F-“ bacterium (lacking the F- plasmid). DNA from the “donor” can be
transferred to the “recipient”. After the conjugation, the former “recipient”, now
containing also an F-plasmid has become a “donor” as well.
The process of conjugation is illustrated below in figure 1:
Figure 1:
The F-plasmid has also the capability to integrate into the bacterial genome. The
bacterium is then called “Hfr” (High frequency of recombination). Like an F+ cell, an Hfr
cell functions as a donor during conjugation: DNA replication is initiated at a specific
point on the integrated F factor DNA. From that point, a single strand of the F factor
DNA moves into the F- partner, dragging along adjacent DNA. For an illustration, see
figure 2 below.
Figure 2:
© B. Woelker, 2008
Conjugation is a form of horizontal gene transfer. Mutation and subsequent binary fission
would be an example of vertical gene transfer.
Conjugation is a major reason for the emergence of antibiotic resistance strains.
Antibiotic resistance genes, which are usually genes that code for an enzyme that can
break down the antibiotic, can be transferred from one bacterium to another by
conjugation. Gene transfer is even possible between different strains of bacteria!
© B. Woelker, 2008