Download M220 Lecture 14 - Napa Valley College

M220 Lecture 14
Mutagenic agents (continued)
Note that when bacterial cells are exposed to U.V. radiation adjacent thymines are unnaturally bonded
to create thymine-thymine dimers (or just thymine dimers). To combat the effect of the U.V. light, many
species possess an enzyme induced by visible light which will cleave or break the covalent bonds joining
these dimers. In addition to this method of repair, a dark repair mechanism exists. In this process an
enzyme first excises the dimer. A DNA polymerase (second enzyme) then inserts new thymines
opposite to the existing adenines to reform the naturally bonded DNA. Too much U.V. radiation can
prevent repair. U.V. light is most damaging at wavelengths between 260 and 270 nanometer.
The above mentioned mutagens will cause an increased rate of mutation for all genes in the genome.
These are general mutagens. However, the mutation rate for different genes can be different. Genes
will have different lengths and configurations increasing or decreasing their susceptibilities.
Genes will mutate independently of each other. The chance that a single cell will have 2 separate
mutations is equal to the product of the single rates. For example let’s say that a cells chance for having
a gene that allows it to be resistant to penicillin is 10-9. Let’s also establish that a cells chance for having
another mutant gene that allow for resistance to streptomycin is 10-5. The chance that a single cell will
have both resistant genes is the product of the individual rates or 10-9 X 10-5 which equals 10-14.
Selecting Mutant Cells
A. Direct Selection
Grow organisms in the presence of an antibiotic. Those that survive will have the mutant gene
that allows those cells to live in the presence of the antibiotic. The word prototroph refers to
the original cells that do not have the mutant gene. The prototroph population is also referred
to as the wild type or normal or parent group of cells. The selected cells growing in the
presence of the antibiotic would represent the mutant population.
B. Indirect Selection
1. Prototroph-grows on a minimal media. Growing cells are susceptible to penicillin
2. Auxotroph-the mutant cells. Auxotrophs are mutants that have additional nutrient
requirements. The auxotrophs will require additional amino acids for growth on minimal
media.
Experimental Procedure for Auxotroph Selection
a. Expose the prototroph population to UV light (intermediate dose).
1. Kills some
2. Mutates some (shocked survivors)
3. Some unaffected (shocked survivors)
b. Grow the shocked survivors on a nutritionally complete media to allow for recovery
c. To separate out the shocked survivors, place them on a minimal media that contains
penicillin.
d.
e.
f.
g.
The prototrophs will grow and then be killed by the penicillin.
The auxotrophs cannot grow on a minimal media. They sit on media and are not killed.
Transfer the cells to a complete media. All auxotrophs grow.
Use replica plating technique to separate the different auxotrophs.
The Ames test makes use of Salmonella histidine auxotrophs to efficiently screen for chemical
carcinogens. Expose these auxotrophs to a suspected carcinogen and look for the presence of the
original Salmonella prototroph. The prototroph does not need added histidine for growth and will grow
on a minimal media. Growth and appearance of the prototroph indicates that the auxotroph
experienced a reversion or a back mutation when exposed to the suspected carcinogen. There is a 90%
chance that this mutagenic agent is carcinogenic. It is deemed a suspected carcinogen.
Genetic Recombination-the exchange of genes between two DNA molecules to form new combinations
of genes on a chromosome. This process creates diversity like mutation and therefore also drives
evolution. Mutation however, only involves a single gene. There are 3 general methods of genetic
recombination available to bacteria. They are transformation, conjugation and transduction.
I.
Transformation
A. Historical perspective-In 1928 Frederick Griffith (English physician) was doing work on
what made pneumococcus (Streptococcus pneumoniae) pathogenic. He found two
different types.
1. Type III(S)-virulent (encapsulated), resisted phagocytosis, smooth colonies, killed
mice.
2. Type II(R)-avirulent ( no capsule), rough colonies, mice survived.
Griffith set up an experiment to see if capsular material killed the mice.
1.
2.
3.
Type III(S) killed the mice.
Type II(R) had no effect on the mice.
Heat killed Type III(S) had no effect on mice.
If the capsule itself was toxic, then the heat killed cells with the capsular material should
have caused disease. Living cells plus the capsule are needed for disease. Not just the
capsule. To continue the experiment the following mixture was used.
4. Mixture of Heat killed Type III(S) and living Type II(R)-this killed the mice.
Were the dead Type III(S) brought back to life? No! Were the living Type II(R) somehow
transformed into the living Type III(S)? Yes! The organism cultured from the dead mice
were indeed living Type III(S).
In 1933 a technique for obtaining cell-free extracts (CFE) was developed. Using this
technique, cells could be broken open and its contents could be spewed out. To
continue experimentation the following procedures were used.