BIO282 Lecture Notes - Lecture 32: Plasmid, System 6, Replica Plating
Learning objectives
By the end of the lecture you should:
1. Realise the effect of a mutation on the cell and how this can provide an understanding
of gene function
2. Have an understanding of the principles of forward genetics
3. Forward genetics-Know how a transposon can be used to study gene function
4. Have an understanding of the principles of reverse genetics
5. Reverse genetics-Understand targeted gene inactivation
Mutation effect
• A forward mutation changes wild-type genotype into a new variation.
• A reverse mutation has the opposite effect and is designated reversion.
• Restoration of the wild-type genotype and phenotype
• i.e. if the mutation is lethal, the cell often reverts the mutation back to normal
• When a mutation alters an organisms phenotypic characters it is said to be a mutant or
variant
• Some mutations are conditionally lethal. These mutations affect the cell only under
defined conditions.
• Mutations may be detrimental (deleterious or lethal) or beneficial
Consider Sickle Cell Anaemia
• Pyrimidine for a purine = transversion mutation
• A single point mutation
• Now we have an amino acid shift from glutamine to valine
Effects at cellular level
• when deprived of oxygen the cells become sickle shaped and can impede
blood flow
Negative effects at the whole organism level
• pain and fatigue.
Positive effects at the whole organism level
• resistant to malaria. The parasites causing malaria are killed inside sickle-
shaped blood cells.
• There is both a deleterious effect and beneficial effect from the single point mutation
Secondary mutations
Sometimes secondary mutations occur
• as a result of the first mutation causing another mutation
Some will not affect the phenotypic expression of another
• Independent
• Two changes will occur
• One mutation causing one phenotype and the other mutation causing
another
Some will affect the phenotypic expression of another.
• These are called suppressor mutations or simply suppressors
• Suppressors may be:
• extragenic (the second mutation is in a different gene to the gene
containing the first mutation), or
Extra (from without)
Genic (from the gene)
• intragenic (the second mutation is in the same gene that the first
mutation is found in)
Intra (from within)
Genic (from the gene)
• The tRNA molecule has its classic anti-codon which is specified for glycine
• The tRNA is charged with glycine
• When it finds that codon, it'll take the glycine into the ribosome and the glycine is
attached onto the existing proteins in the ribosomes
• If you get a mutation
• That DNA sequence will produce and RNA molecule
• e.g. the A base has been substituted for the original G base
• A and G are both purines so this has to be a transition type mutation
• This impacts the cell significantly because it changed the codon from glycine to
arginine
• This may or may not have an impact, but it's still a mutation
• If that particular protein is essential a secondary mutation can result and supress
any phenotype that comes as a result of that mutation
• It's trying to restore the wild-type phenotype
• You can not only get a reversion (A changed back to G) but you can also get a
suppressor mutation (another mutation occurring somewhere else)
• In this case the suppressor mutation is located in the anti-codon sequence of
the tRNA molecule which has glycine on it
• In the sequence, instead of being 5' U C C, it is now 5' UCU , it is still the
tRNA charged with glycine, but it can recognise the arginine codon
(different) and in its place put the glycine back in. restoring the wild type
protein
Document Summary
These mutations affect the cell only under defined conditions: mutations may be detrimental (deleterious or lethal) or beneficial. Consider sickle cell anaemia: pyrimidine for a purine = transversion mutation, a single point mutation, now we have an amino acid shift from glutamine to valine. Effects at cellular level: when deprived of oxygen the cells become sickle shaped and can impede blood flow. Negative effects at the whole organism level: pain and fatigue. Positive effects at the whole organism level: resistant to malaria. The parasites causing malaria are killed inside sickle- shaped blood cells: there is both a deleterious effect and beneficial effect from the single point mutation. Sometimes secondary mutations occur: as a result of the first mutation causing another mutation. Some will not affect the phenotypic expression of another. Independent: two changes will occur, one mutation causing one phenotype and the other mutation causing another.
