Genetics
Dr. Shakuntala Pillai
Credit 2 Model genetic systems; Mutation, mutagenesis
and mutant selection; Mendelian inheritance;
Alleles, Complementation.
Reference:
Snustad/ Prescott (For this entire ppt)
Terminologies: auxotroph, prototroph, revertant, Ames test, carcinogens, minimal medium, replica
plating, conditional lethal mutants
What we have studied till now….?
• Types of mutation: molecular changes in DNA
• Types of Mutation: where they occur
• Types of mutation: how they occur
• How the genetic codon resists mutation? (that’s why it is conserved
from bacteria to human)
Mutations
• Types with and without frameshift
• Types of substitution mutation based on consequence (silent, missense, nonsense)
• Types of substitution mutation based on change in nucleotide(transition and transversion)
• Types based on tissue or cell type (germline & somatic)
• Types of mutation based on the cause (Spontaneous and induced)
• Mutation due to insertion of large fragment of DNA
• Cis and trans mutation
• Mutation and alleles
• Reversion of mutation
• Complementation
• Hotspots of mutation
• Where we use mutation in research?
New and old terms that we already know..
• A mutation is a heritable change in the base sequence of that
genome, that is, a change that is passed from the mother cell to
progeny cells.
• A mutant by definition differs from its parental strain in its genotype,
the nucleotide sequence of the genome.
• The observable properties of the mutant—its phenotype—may also
be altered relative to its parent. This altered phenotype is called a
mutant phenotype.
• Note: that’s why we have seen in previous classes that in some
mutations few rounds of replication are required for bringing the
genetic change in both the chromosomes.
Conditional Lethal Mutants in bacteria
These mutants can behave normal and can behave like mutant under
given circumstances.
Mutations that are lethal in one environment, the restrictive condition,
but are viable in a second environment, the permissive condition.
(1) Auxotrophic mutants,
(2) Temperature sensitive mutants,
(3) Suppressor sensitive mutants (not discussed in the class)
Auxotrophic Mutants and prototrophs
• Auxotrophs are mutants that are unable to synthesize an essential
metabolite (amino acid, purine, pyrimidine, vitamin, etc) that is
synthesized by wild-type or prototrophic organisms of the same
species.
• The auxotrophs will grow and reproduce when the metabolite is
supplied in the medium (the permissive condition); they will not grow
when the essential metabolite is absent (the restrictive condition).
How will you screen or identify auxotrophic
mutants?
Step 1 :
In a flask, culture of bacteria.
Exposed to mutagen eg. UV
Step 2:
Grow the bacteria in complete medium
permissive condition
Step 3:
Replica plate(‘copy +paste’) all the
bacteria
Step4:
Bacteria that grow in complete medium
but fail to grow in minimal medium are
auxotroph
permissive condition the restrictive condition
Single Bacteria multiply to form visible
colonies on solid nutrient medium.
If you have so many bacterial colonies on a
plate, it becomes difficult to transfer each
of them from permissive to restrictive
medium plates.
Replica plate technique
• Identify the colonies that are missing in
the restricted (minimal) media.
Temperature sensitive mutants
• Temperature-sensitive(Ts) mutants will grow at one temperature
(permissive condition) but not at another (restrictive conditions).
• heat-sensitive or cold-sensitive.
• Guess where the mutation could be in such mutant bacteria?
• The temperature sensitivity usually results from the increased heat or
cold lability of the mutant gene product—for example, an enzyme
that is active at low temperature but partially or totally inactive at
higher temperatures.
Methods for Testing samples for carcinogens
makes use of auxotrophic mutants
• Mutagenic agents are also carcinogens;
• they induce changes in DNA that may led to cancer
Screening Chemicals for Mutagenicity:
The Ames Test
• Ames and coworkers constructed auxotrophic strains of the bacterium
Salmonella typhimurium carrying various types of mutations(~8
regions)—transitions, transversions, and frameshifts—in the genes
required for the biosynthesis of the amino acid histidine.
• The reversion of these auxotrophic mutants to prototrophy
• comparing the frequency of reversion in its presence with the
spontaneous reversion frequency
• Ames test is used to test pharmaceuticals, agrochemicals, food additives,
cosmetics and personal hygiene products etc.
Ame’s Test to
identify mutagens
Critical thinking
[Link] in Ames test we look for auxotroph to prototroph conversion?
Why not from WT to auxotroph?
• The answer will make you realise how important are auxotrophic
mutants or conditional lethal mutants…Also now you know that
bacterial mutants can be used for human benefits like testing
carcinogenicity of any chemical.
2. Mutagens that causes mutation can also cause reversion of
mutation!!