Mutational analyses

The power of mutational analysis is that after the investigator has defined their screen or selection, the organism provides the mutations without regards to the investigator's preconceptions.

Genetic analysis can often reveals unexpected components of a process.

At the same time, it is worth remembering that not all genes are equally mutable and a particular gene may be involved in many processes.

Many genes involved in a process may be difficult or impossible to recognize by simple genetic methods.

Mutations arise spontaneously, due to mistakes in DNA replication, chemical or radiation induced damage to DNA. Spontaneous mutations are useful, since it is likely that only a single gene has been altered.

It may be difficult, however, to accumulate enough spontaneous mutations. To get sufficient numbers it is often necessary to induce them.

MULLER discusses the induction and type so mutations

Mutations can be induced using radiation, e.g. UV light, X-rays, gamma-rays, or by exposure to mutagenic chemicals -- all act by changing the structure of the genetic material, DNA.

In our experiments, we will use UV light to mutagenized a culture of E. coli.

Making Mutations

UV mutagenesis primarily leads to the formation of thymidine dimers within DNA and requires the cells contain an "SOS" repair system, which E. coli does.

CAREFUL: Since we humans also have an SOS-like system, so exposure to UV light generates mutations in our cells and this is to be avoided.

Our experiment will be will be carried out in two steps.

First we will calibrate a standard 15 Watt "germicidal" UV lamp, which can be purchased from most electrical supply companies.

To determine the UV output of our system, we will use a bioassay, the killing of E. coli cells.

Start by growing up an overnight culture of E. coli in LB. In the morning, dilute the cultures into fresh LB and grown to a density of ~ cells/ml.

Components in LB absorb UV light, and so reduce the effective dose of radiation.

We will therefore 'wash' the bacteria by centrifugation and resuspend them in a UV-transparent medium (phosphate-buffered saline or PBS).

Based on many studies, it is known that a UV flux 50 J/m^2 will reduce the viability of E. coli by 60%

Optimal mutagenesis occurs when the level of bacteria that survive exposure to UV is ~1%.

Using your lamp, how long an exposure will you need?

Making and finding a specific type of mutation

Having calibrated our UV lamp, we can determine whether we can use UV to generate mutations that specifically enable bacteria to grown in the presence of the antibiotic rifampin.

Most of the current generation of antibiotics are derivatives of natural products.

Rifampin, for example, is a derivative of rifampicin, made by the eubacterium Streptomyces mediterranei.

Rifampin can kill a wide range of bacteria by binding to and inhibiting the activity of their DNA-dependent, RNA polymerase, an essential enzyme.

Based on your previous estimate of UV flux, we will begin by exposing the cells to a dose of UV light sufficient to kill between 99% and 99.9% of the cells.

What is the frequency of rifampicin-resistant mutants in your cultures?
Is there an increase in the number of rifR mutants as UV dose increases?
Is the relationship a linear function of dose?
What other experiments would you like to do to answer this question?
When you isolate rif- mutants how can you determine whether the mutants are related (that is share a common ancestor) or independent.
How could you insure that you are working only with independent mutations?

revised 9 July 2003