Dissection of Gene Function part I: Forward Genetics Flashcards
We have two principal approaches to learn about gene function:
Forward and Reverse Genetics
Two strategies which are based on inheritable changes:
LIST AND DEFINE BOTH
- Forward Genetics - Look for mutant phenotypes of interest followed by molecular analysis of mutants
- Reverse Genetics
– Mutate a known
gene - infer function by change in phenotype compared to wt
phenocopying
an environmental induced, non-heriditary phenotype of one individual which is identical to the genotype-determined phenotype of another individual.
Phenocopying causes
disruptions that are (usually) not inherited.
Gene function and study of Genetics
Genetics enables us to study abnormal gene function in mutants to inform us about normal gene function.
Forward Genetics can be done in two principal ways:
- Forward Genetics analyses heritable phenotype of a mutant at the genetic level then perform a molecular analysis of the mutant.
1 * Scenario 1: Survey the genome for ALL genes that contribute to a particular biological process
– Start with wild type genome
– Mutagenize randomly a large population of wt genomes
– Systematic survey for mutations that share the desired phenotype suggesting that these mutation affects the process under investigation.
– Perform molecular analysis of all these mutants
- Scenario 2: Mutated gene causing a phenotype is known. More mutations in that gene should be investigated to better understand that single gene’s function
– Collection of mutants is tested for mutations in that particular gene followed by
cloning and sequencing
– Correlate mutations with observed phenotypic changes (e.g. severity)
Forward Genetics:
Scenario 1: Survey the genome for ALL genes that contribute to a particular biological process = 4
– Start with wild type genome
– Mutagenize randomly a large population of wt genomes
– Systematic survey for mutations that share the desired phenotype suggesting that these mutation affects the process under investigation.
– Perform molecular analysis of all these mutants
Scenario 2: Mutated gene causing a phenotype is known.
More mutations in that gene should be investigated to better understand that single gene’s function =2
– Collection of mutants is tested for mutations in that particular gene followed by
cloning and sequencing
– Correlate mutations with observed phenotypic changes (e.g. severity)
Choice of mutagens: 2
- Either use naturally occurring mutants
- Or find a mutagen that induces mutations
– Transposons
* e.g. maize Ac/Ds transposons
– Transform one plant with an Ac element, another with a Ds element
» Crossing of the two transgenic plants activates Ds which transposes and interrupts genes
» Outcrossing Ac removes transposase ➔ Ds element trapped and
tags the mutation ➔ easy to isolate the mutated gene
– Chemical mutagenesis:
- e.g. using EMS (Ethyl methanesulfonate), MMS (Methyl methanesulfonate),
ENU (Ethyl-nitrosourea) ➔ requires uptake of mutagen into the cell
– Radiation mutagenesis
- UV: good for microbes
- X rays, gamma rays: cause large-scale changes such as intragenic deletions
Choice of mutagens: Transposons
Transposons
* e.g. maize Ac/Ds transposons
– Transform one plant with an Ac element, another with a Ds element
» Crossing of the two transgenic plants activates Ds which transposes
and interrupts genes
» Outcrossing Ac removes transposase ➔ Ds element trapped and tags the mutation ➔ easy to isolate the mutated gene
Choice of mutagens: Chemical mutagenesis
Chemical mutagenesis:
- e.g. using EMS (Ethyl methanesulfonate), MMS (Methyl methanesulfonate),
ENU (Ethyl-nitrosourea) ➔ requires uptake of mutagen into the cell
Choice of mutagens: Radiation mutagenesis.
– Radiation mutagenesis
- UV: good for microbes
- X rays, gamma rays: cause large-scale changes such as intragenic deletions
Chemical mutagenesis using EMS
1* mutagenic, teratogenic, and possibly carcinogenic
2 * Alkylates guanine
3 * During replication, alkylated guanine will base pair with T instead of C
4 * After another round of replication, the base pair
will change to a T-A base pair
➔ transitional point mutation from C-G to T-A
Ionizing or non-ionizing radiation mutagenesis
Define and Explain them:
- IONIZING RADIATION was the first mutagen that efficiently and reproducibly induced
mutations in a multicellular organism.
– X rays, gamma rays (γ), beta particle radiation (β), and alpha particle (α)
radiation
– X-rays and gamma rays cause single strand breaks
- NON-IONIZING RADIATION
– UV radiation, like that in sunlight, is non-ionizing.
Causes thymine dimers. If not repaired lead to errors during replication
Mutation rate versus Mutation dose:
EXPLAIN Mutation rate: + 3
- Mutation rate: frequency of new mutations in a single gene or organism.
– Ensure that:
* mutagen produces sufficient mutations to enable recovery of desired
mutations
- mutagen does not produce more than one mutation per genome ➔ multiple mutations make genetic analysis difficult or kill the cell
– Usually: a mutagen dose that leads to 50% survival rate is aimed for.
Mutation rate versus Mutation dose:
DEFINE + WHY DIFFICULT OT ACHIEVE 3
Ideally try for SATURATION MUTAGENESIS:
all genes in the genome that confer a specific phenotype are mutated.
Difficult to achieve because:
1 - Gene size determines probability of mutation in that gene
2 - Mutations are often pleiotropic ➔ they have numerous effects on phenotype
3 - Mutations can be severe: death, infertility
Mutations are rare:
frequency ≤ 10-5
➔ Effective assay systems needed to recover desired mutants
Assay Systems for Forward Genetics:
Genetic Selection 4
1 – Killing or inhibiting wild types and non desired mutants, mutants survive
2 – Easy to do
3 – Advantage: only desired mutant survives
4 – Often used for microbes – supplement growth medium with a selective agent
Selective agents may be: 5
1 * Specific nutrient toxic for non-mutant
2 * Growth on absence of a specific nutrient
3 * Inhibitors
4 * Pathogens
5 * Antibiotics
genetic selection vs genetic screening
Mutagen into Bacteria growing in liquid culture.
to
- Genetic Selection:
Individuals lacking phenotype of interests are killed.
- Individual with mutant phenotype of interest survives
or
2. Genetic Screen
- Numerous individuals survive
- phenotype of each survivor must be examined
—individual with mutant phenotype of interest is found.
Prototroph: 2
1 – Organism or cell capable of synthesizing all its metabolites from inorganic material, requiring no organic nutrients
2 – Can grow on minimal
medium
Autotroph: 2
– Can synthesize its food from inorganic substances
– Can grow on minimal medium but requires heat or light as energy source
Example of a Forward Genetic Selections
– selection of auxotroph mutants
Auxotroph:
– Mutant organism, that requires the addition of a particular organic compound they cannot synthesize
– Do not grow in Minimal medium, require Opposite of autotroph – self-feeding
Example of a Forward Genetic Selections
Selection of auxotroph mutants in filamentous Fungi using filtration enrichment.
1 * Prototrophic wt filamentous fungi cells form ‘fuzzy balls’ when grown on liquid minimal medium
2 * Auxotrophic mutant cells do not grow
3 * Wild type is filtered off and discarded
4 * Mutant cells are in the filtrate and can be grown on minimal medium supplemented with the required organic compound
– e.g if leucin is the requirement compound then leucin auxotrophs will grow
5 * Can do this with a supplementation of a variety of organic compounds to select for different auxotrophies
* ➔ investigate metabolic pathways
Example of a Forward Genetic Selections - simplified 4
- Minimial medium:
- Prototrophic wild types
- nutrional mutants (auxotrophs) - Prototrophs held by filter
- Auxotrophs pass through
- Onto plate
Auxotrophs can grow…Leucine-containing medium
