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March 24 2010
Lecture 31: Genetic Dissection II - Forward Genetic Analysis
Forward vs. Reverse Genetics:
 Reverse Genetics:
 Also called modern genetics
 From gene/DNA sequence to function(s)
 Analyze mutants of a specific gene to understand it’s functions
 Forward Genetics
 From mutant phenotype to identification of the underlying gene function(s)
 Analyze mutants defective in the process of interest to understand the
causal gene(s)
 Clone gene(s) identified to determine molecular role(s)
 Also called classical genetics
 Forward and reverse genetics are complementary
 Sometimes knocking out genes my give subtle/no phenotype- hard to distinguish
 Lack of phenotype may be because multiple genes are responsible for one
phenotype (functional/genetic redundancy), so another gene function
compensates for the mutated gene
 In case of genetic redundancy one may need to knock out multiple genes
to se a phenotype and/or determine function
 In situations like this reverse genetics can be easier and faster
Genetic Dissection: Discover mutants defective in specific biological processes to
identify genes and proteins involved.
 Genetic Screens: A search through natural or mutagenized population to identify
mutants unable to perform the process of interest.
 Affected gene(s) are required for ad/or involved in that process
 Examples of screens: Morphological, behavioural, biochemical
 Mutation is a rare event and mutagens increase mutation frequency and ease the
search for genes involved in biological pathways
 Mutagens: increase the frequency of mutations by various means
 Chemical Mutagens:
 Point mutations, affect proteins through truncation or amino acid change
 Can cause frame shift mutations, or premature stop codonsnonsense mutations
 Eg. Ethylmethane sulfonate
 Radiation
 Small or large deletions of DNA, chromosomal breakage and
rearrangements, depending on type and dose of radiation
 Eg. X-Rays and fast neutrons
 Insertional Mutagens
 Insert randomly into the genome and by chance may end up in a gene, or a
regulatory region of a gene, disrupting their expression
 Inserted DNA is known, and therefore acts as a tag to the mutation, and
therefore the gene involved in the mutant phenotype
 Can be engineered/ tailored to drive this process
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 Eg: Transposable elements, T-DNA in plants, P-elements in Drosophila
 Inducing mutations with a mutagen is a “balancing act” because you want to have
enough mutations to merit studying, but you don’t want them to get confused with
multiple mutations in a single organisms, and you don’t want to kill the organism
Genetic Screens: Selection versus Screening
 Selection: Out of a collection of mutants, engineered the environment/ other
conditions such that only the desired mutants survive
 Kill or inhibit growth of non-desired mutants
 Easy to screen large populations
 Limited to specific genes
 Example: Auxotrophic mutants of fungi
 20 possibly mutant colonies grown in 4 different conditions
 Controls:
 Positive: Minimal media + Leucine+ Adenine
 All colonies can live on this
 Negative: Minimal media (carbon compounds, water etc.)
 Only colonies that can synthesize Leucine and
Adenine survive on this control
 Colony 2:
 Grows in positive control, not negative control
 Grows in presence of adenine and minimal media, but not
leucine and minimal media
 Therefore is a mutant that cannot make it’s own adenine
 Colony 14 is the inverse of this
 This is an example of comparing growth conditions to determine
requirements
 Screening: Analyze all the individuals of a population and separate them into two
categories- interesting and non-interesting
 More laborious because you have to analyze all individuals in a population
 Less biased
 Allows identification of unexpected phenotypes, yielding a greater range
of phenotypes
 Example: Cell cycle mutants in budding and fission yeasts
 Looking for something that induce death
 Using temperature sensitive alleles to study lethal mutations
 At permissive temperatures both mutant and wild type are
functional: no phenotype
 As temperature is increased to restrictive temperature, the
mutant protein does not function: mutant phenotype
 Budding Yeast:
 Normal yeast bud, and the buds grows then separates from
parent , once it is large enough, by mitosis
 Mutations at restrictive temperature:
 Premature induction into meiosis generating cells
that are too smalls
 Cells that are unable to produce buds generating
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large mutants that have replicated DNA
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Fission Yeast:
 Elongates and separates
 Mutations at restrictive temperatures:
 Defective DNA segregation- cell is unable to divide
 Elongated DNA that cannot divide
 This mutant analysis led to the discovery of cdc genes
Selection and Screening are complementary and chose on nature of results that you
are looking for
Genetic Analysis of Mutants: Once we have identified a collections of mutants- what do
we do with them?
 Detailed Phenotypic Analysis
 What exactly is altered in the mutant?
 Morphology, biochemistry, behaviour
 Make sure we all know ALL of the effects, not just the main ones
 Genetic Analysis:
 Classic
 Dominant? Recessive?
 How many genes affect the individual mutant phenotype?
 How many different genes have been identified in the screen?
 Gene interactions
 How does the environment affect what we are observing?
 Gene cloning
 Genetic Analysis:
1. Is the mutant allele dominant or recessive to the wild type character?
 Cross each mutant with the wild type and observe the phenotypes of F1
and F2 progeny
 F1:
 If all wild type, cross again
 If all mutant than the mutation is dominant
 F2:
 3:1 rations of Wild Type : Mutation means one gene and
recessive
 Other ratios- other conclusions
 Recessive mutations
 Generally loss of function mutations: null (amorphic) or decreased
function (hypomorphic)
 Recessive to wild type because are haploinsufficient
 Dominant mutations
 Both loss of function and haploinsufficient
 Gain of function (too much) hypermorphic
 Novel function (neomorphic)
2. How many genes are affected in each individual mutant?
 Is the defective phenotype of a particular mutant the result of a mutation in a single or
multiple genes?
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Mutagenesis may cause multiple mutations in the same individual
Cross each mutant to wild type twice and analyze the second generations (F2)
 Analyze the segregations rations of mutant versus wild type phenotypes
3. How many genes are isolated in the screen?
 Complementation test
 Cross mutants with each other
 If mutants are in the same gene (allelic) they will NOT complement
 You will still have the mutant phenotype
 If the mutations are on different genes they will complement
 You will have wild type phenotype
 Allows us to categorize mutants into complementation groups
4. How do the different genes isolated interact with each other in the wild type
phenotype?
 Double Mutant Analysis
 Cross the mutants with each other and analyze the double mutant progeny
 Combination of parental phenotypes: additive
 Two things may be happening and in parallel
 Looks worse than either parent
 Synergistic phenotype: two things happening in parallel with the same
final result added on each other give a greater final effect
 Look like only one of the parents:
 Epistatic Phenotype: the gene from the parent of the phenotype shown
acts first in a genetic pathways
Gene Mapping: locating a gene with a genome
 Recombination-based maps:
 Relative position of loci of the genes for which mutant alleles have been found.
Determined on the basis of frequency of recombination at meiosis
 Units: Recombination frequency (RF), map unit (m.u.) centi-Morgan (cM)
 Physical Maps
 Using the map of the genomic DNA from sequencing data
 Includes genes, spacers and regulatory sequences, non-coding sequences etc.
 Unit: kilo base (kb) - physical unity
 Need both because each gives different information
 Can now look for conservation
 May be the first step to cloning a gene based on its “map position” - Positional
Cloning
 Looking at progressively smaller fragments until the gene of interest is isolated
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