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
Testing Neurospora crassa strains for auxotrophy using forward genetic selection
- N. crassa: model organism, a filamentous fungi, type of red bread
mold
2.* 20 progeny from a cross adleu+ x ad+
leu–inoculated on minimal medium (Min) or
minimal medium with adenine (Ad) or leucin (Leu) or both
3 * ad mutants cannot produce adenine, leu mutants cannot produce leucine
4 * Look for colonies growing on the different media to determine which auxotropy they have and to isolate a double auxotroph.:
Forward Genetic Selections can also be done to identify mutants in animal behaviour
PHOTOTAXIS MUTANT
Wild type Drosophila shows
PHOTOTAXIS: orientates itself and flies towards light in the T-maze
PHOTOTAXIS MUTANT screen identifies Drosophila mutants that lost the ability to fly towards light ➔ flies appear at the light and dark side of the Tmaze with equal probability
Assay Systems for Forward Genetics:
Genetic Screens what is it?
disadvantage/advanatge
Genetic Screen
– Identify desired phenotype
– More work
– Advantage: a greater range of phenotypes
can be looked for
Genetic screens can be used to dissect any biological process = 4
1 – Development
2 – cell cycle
3 – signal transduction
4 – metabolism etc
Genomics has facilitated Forward Genetic screening
- application of high-throughput approaches to
Genetics
Morphogenesis:
- Morphogenesis: development of form
Mutagenize large population of haploid cells and plate those
– Haploid ➔ even recessive mutations can be observed, cannot be covered by a dominant wt allele
Forward Genetic Screens to identify Morphogenesis mutants in Neurospora = 3
1* Mutagenize large population of haploid cells
and plate those
– Haploid ➔ even recessive mutations can be observed, cannot be covered by a dominant wt allele
2 * Identify changes in colony morphology
3 * ➔ hundreds of loci identified that are involved in tip growth and branching of the filamentous fungus.
– mutations found in many cytoskeletal genes such as actin, dynactin and dynein
yeast
single cell eukaryotic fungi
- Saccharomyces cerevisiae – budding yeast (beer, bread) =2
– Outgrowth from the parent produces a bud
– first complete DNA sequence of a eukaryotic
genome (1996)
Schizosaccharomyces pombe – fission yeast
– Fission: results in two identical individuals by
splitting
TAPOYG - The Awesome Power Of Yeast Genetics:
Both are model eukaryotic organisms for genetics and molecular biology because
– Many genes in yeast and mammals encode
very similar proteins
– Investigating yeast mutants has many valuable outcomes for research in other eukaryotic organisms, including humans
Conditional heat sensitive
mutants
1 * Temperature-sensitive mutants are important tools for understanding the role of
essential gene(s).
2 * Often, they result from amino acid changes due to mutations of genes.
3 * Restrictive temperature: high growth temperature at which the mutation causes the protein to malfunction or not function at all ➔ often deleterious ➔ mutant phenotype
4 * Permissive temperature: lower temperatures at which the mutation does not impact on protein function ➔ wt phenotype
What happens in Permissive Temperatures? 5
- Unfolded polypeptide
- Folding
- Binding to cognate partner
- Active protein
- WT phenotype
What happens in Restrictive temperatures?
Unfolded polypeptide
- Unfolding or misfolding
- proteolysis or Aggregation
- Aggregation -> Decreased amount of active protein
- TS phenotype
OR
1. folded and stable
2. Low affinity for partner
3. TS phenotype
Visual identification of mutants with abnormal celldivision cycle - cdc mutants
1 – Expectation: many cdc mutants are lethal
2– Screen for conditional heat sensitive mutants:
* Nobel prize for Leland Hartwell and Paul Nurse
Mutations block the mitotic cell cycle at different,
specific points 4
(a) Abnormal mitosis - DNA segregates irregularly along spindle
(b) haploids enter meiosis
(c) mutants elongate without dividing
(d) Mutants arrest without budding
Forward Genetic Screens to dissect the Cell Cycle in Yeast using heat sensitive screens
What identified? Comparative genomics?
- Identified many proteins that regulate and control the
cell cycle progression - Comparative genomics has shown that these same genes are at work in the cell cycle of humans, and that
many of these genes are defective in cancers.
A Visual Forward Genetic Screen for nuclear division
defects in Aspergillus
- A visual screen for altered nuclear division
(screened at lower than normal growth temperature, see yeast) - 3 classes of heat sensitive mutants showed key players in cell division and growth:
– nimA - never in mitosis
* codes for a kinase
– bimC - blocked in mitosis
* codes for kinesin, a motor protein that moves organelles on cytoskeleton
– nudA - nuclear distribution
* a dynein subunit, also moves organelle
Aspergillus
is a filamentous fungus, genetic
model organism
A Visual Forward Genetic Screen for Dissection of Development in Zebrafish
- Small, develop rapidly, and produce many offspring ➔ good model for vertebrate development
- Transparent embryos ➔ easy to observe abnormalities in early development
- Male take up a chemical mutagen ENU (Ethyl-nitrosourea)* via tank water
– mutagenised males produce BASE-SUBSTITUTED sperm with many different mutations - *****F1 contains one normal chromosome (mother) and one mutagenised (father)
– F1 males x WT females - Offspring interbreed to reveal recessive phenotypes
Forward Genetic Screens made easier
using haploid Zebrafish =4
1 * Speed up zebrafish mutant screen by producing haploid offspring
2 * Achieved by UV irradiation of F1 males
– destroys sperm nuclei (heavily
mutagenized) so they cannot
contribute sperm genomes to the zygote
but can still fertilize egg
3 * Offspring don’t develop to adults but can look for mutants in early development
4 * Half of the offspring carries newly induced mutation inherited from the
female parent
Forward Genetic Screen to Identify Enhancer elements in eukaryotic genomes using enhancer trapping
1 * Eukaryotic enhancers (DNA regulatory sequences) act as enhancers of transcription of genes whose transcription start site is nearby
2 * Hunting for enhancer sequences using enhancer trap: randomly insert a transgenic reporter construct (e.g. GFP) into genome ➔ reporter is activated if it inserts near an
enhancer.
3 * Expression of reporter gene due to enhancer activity can be observed
4 * Identify enhancer elements that drive particular gene expression pattern, e.g. developmental, organ specific…
5 * If the enhancer trap is on a transposon ➔ can be mobilised and move to other
genome positions to identify further enhancers
Minimal Promoter - Reporter Gene =
Minimal Promoter - Reporter Gene = No or weak expression
Enhancer + Minimal Promoter to Reporter gene = strong GENE EXPRESSION
Forward Genetic Screen to Identify Enhancer elements
involved in Drosophila = 2
- Genes A and B in the Drosophila genome are regulated by enhancer elements in a developmental
fashion. - Insertion of an enhancer trap leads to activation of a
reporter gene by the enhancers. The activation is
development specific
