Topic D - Introduction to Genetic Analysis

Non-Conditional Mutation

Shows the mutant phenotype under all conditions

Ex; Pyocyanin Production - Mutant in 1 gene involved in pyocyanin production

Mutants are defective at all temperatures and on all media

Can either be a null phenotype (complete loss of WT phenotype) or leaky phenotype (partial loss of WT phenotype)

Conditional Mutation

Does not always show the mutant phenotype

-Dependent upon environmental conditions or other mutations

1. Permissive Condition - No mutant phenotype observed (WT phenotype observed)

2. Restrictive Condition - Mutant phenotype observed

Conditional-Lethal Mutation

Conditional mutation in an essential gene, viability depends on growth conditions

-Allows for lethal mutations in haploid organisms (phage, bacteria)

Only way to genetically analyze essential genes in haploid organisms

Permissive and Restrictive - Conditional-Lethal Mutation

Permissive Condition - Bacteria grow, phage able to replicate (acts like WT)

Restrictive Condition - Bacteria do not grow/phage cannot replicate (displays mutant phenotype)

Conditional Mutation Examples

1. Auxotrophs

2. Temperature-Sensitive

3. Suppressor Sensitive

Auxotrophic Mutants

Lack the ability to synthesize a compound required for growth, always conditional-lethal

-Usually due to a mutation that inactivates a metabolic pathway

1. Restrictive Condition - growth on minimal media

2. Permissive Condition - growth on supplemented/rich medium

-Supplies the nutrient the mutant cannot make itself

Non-Lethal Temperature Sensitive

Phenotype is different at different temperatures

-WT phenotype at lower temperatures (ex; 30C)

-Mutant phenotype at higher temperature (ex; 37C)

Product of a mutated gene is unstable at higher temperature due to a missense mutation (folds at low temp = function, can’t fold at high temp = non-functional)

Siamese Cats

Siamese cats exhibit temperature-sensitive albinism

-Homozygous for ts mutation in Tyrosinase, which forms the melanin pigment

Conditional-Lethal Temperature Sensitive

Temperature sensitive mutation in an essential gene

-At permissive temperature, organism appears WT

-At restrictive temperature, organism is dead

lpxA2 - Conditional-Lethal Temperature Sensitive

lpxA protein functional at 30C → LPS made → bacterium grows

-lpxA protein non-functional at 42C → LPS not made → bacterium dies

Suppressor Sensitive Mutants

Involves two mutations that genetically interact:

1. Original Mutation (mutation #1)

2. Suppressor Mutation (mutation #2)

-Phenotype from OG mutation is observed in some bacterial strains, but not others, depending on presence of suppressor mutation

-Tend to be information/nonsense suppressors

-May be conditional-lethal if nonsense mutation is in an essential gene

Restrictive Conditions (Suppressor Sensitive Mutants)

Strain contains original mutation, but not the suppressing mutation

-Mutant phenotype caused by original mutation is observed

Permissive Condition (Suppressor Sensitive Mutants)

Strain contains original and suppressor mutations

-Mutant phenotype caused by original mutation is NOT observed

Informational Suppressor

Nonsense mutation by original mutation introduces stop codon in mRNA

-Suppressor mutation → mutation in anticodon of tRNA that can base pair with stop codon

Nonsense Suppressor Strain

Bacterial strain that contains tRNA mutation, causing the stop codon to be read as an amino acid

-Strains may grow poorly:

1. Sometimes misreads the real stop codon = non-stop

2. Can produce too-long proteins that fold incorrectly or are unstable

Permissive Condition - Nonsense Suppressor Strain

Original and suppressor mutations present:

-Has tRNA anticodon mutation

-Full length protein is made

-Bacterium/virus can survive/replicate

Restrictive Condition - Nonsense Suppressor Strain

Only original mutation present:

-No tRNA anticodon mutation

-Functional protein not made because of nonsense mutation

-Bacterium or virus cannot replicate and dies

Classification of Suppressor-Sensitive Mutations

Classified depending on type of nonsense codon that is suppressed

Isolation of Mutants

Performed to identify rare mutants affected in a specific process:

1. Grow culture of organisms

2. Treat with mutagen (or spontaneous)

3. Spread onto solid medium to observe large number of organisms (colonies, plaques, etc)

4. Screen or select

4a (Screen) vs 4B (Select)

Screen - Both mutant and wildtype can grow

Selection - Mutants grow in conditions where WT parent cannot, can find one mutant out of billions of cells

-Easier than screening

Screening

All organisms can grow, both parent and mutants for colonies/plaques

-Identify mutants of interest based on phenotype\

Example: Blue-White Screen, desired phenotype have no B-galactosidase activity

-Functional B-galactosidase → cleaves X-gal → appears blue (WT phenotype)

-Non-functional B-galactosidase → can’t cleave x-gal → appears white (mutant phenotype)

Replica Plating

Used to screen for conditional lethal mutants (can’t select since direct phenotype = death/no growth)

1. Bacteria/phage first plated under permissive conditions

• Mutants will have WT phenotype, so everything will grow

2. Copy of plate is made and grown under restrictive conditions

• Mutants will not grow

3. Compare growth pattern between permissive and restrictive conditions

• Colonies missing on restrictive plate have conditional-lethal mutation

T4 rll Mutants

Contain a conditional-lethal mutation, isolation requires;

-Plate phage on permissive host (E.coli B) and compare plaque size

-Replica plate phage on E.coli B (permissive condition) and E.coli K (restrictive condition)

Selection

Plates under conditions which only desired mutants will grow

-Only works for very specific types of mutations

Example: Host Range Mutants, his- revertants

Host Range Mutant (Bacteria)

Mutant bacteria mixed with WT phage with high MOI

-Some bacteria will contain mutations preventing phages from replicaitng

-Only bacteria with phage-resistant mutation will grow

Host Range Mutant (Phage)

Mix mutant-resistant bacteria with mutated phage

-Phage may contain a mutation allowing to infect resistant bacteria

-Phage kills bacteria → releasing more phage → results in plaque formation

-All phage within plaque contian mutation to infect resistant bacteria (whereas WT can’t)

Only works for certain mutations

his- Revertant

his(-) auxotrophs cannot grow without histidine (can’t grow on minimal media)

-However revertant can grow in minimal media

Therefore, only his+ revertants can grow

Enrichment

Increases the proportion of cells in a population containing a specific desired mutation

-Reduces amounts of effort needed for subsequent screening because your desired mutants are more common in the bacterial population

NOT selection for mutation

Penicillin Enrichment

Penicillin - Antibiotic that blocks formation of crosslinks in peptidoglycan

-Only affects growing, dividing cells

Method of Penicillin Enrichment

1. Mutate bacterial cells

2. Allow all cells to grow under permissive conditions for short time (parents/mutants grow)

3. Transfer to restrictive conditions (parent cells grow, but not mutants)

4. Add penicillin (blocks new peptidoglycan formation in dividing cells)

• Dividing cells will be killed, non-dividing mutants will survive

5. Plate surviving cells on permissive conditions (mutants can grow) without penicillin

• Many of the survive cells contain desired mutations)

Still requires screening to identify mutants

Penicillin Enrichment to Isolate Auxotrophs

-Mutagenized culture in rich medium contains auxotrophic + prototrophic cells

-Suspend in medium minus one nutrient + penicillin, prototrophs grow and 99% are killed by penicillin but auxotrophs cannot grow and are not killed → culture enriched for auxotroph

-Transfer to rich medium, then perform replicate plate to locate auxotrophic colonies on the original plate (rich medium)

Complementation

Tests to determine if two mutations are in the same or different genes

-Proteins synthesized from 2 different DNAs work together in a single cell to produce a WT phenotype

-Progeny are not studied

Recombination

Used to map relative positions of genes/mutations

-Physical exchange of DNA between two different parent DNA molecules

-Progeny have different DNA sequences/phenotypes than both parents (recombinants)

Problems in Genetic Analysis of mutants

Recombination/Complementation require 2 copies of gene being studied in the same cell

-Bacteria/phage are haploid

Solution: Create a pseudodiploid with two copies of the gene in one cell

Types of Pseudodiploid

1. Merodiploid

2. Coinfection

Merodiploid

Used for bacteria

-Second copy of gene introduced on a plasmid

-Bacteria only diploid for those genes on the plasmid

Coinfection

Used for phage

-Infect bacteria with 2 different mutant phage stocks at high MOI

-Both phage infect and replicate at the same time

Complementation Process

1. Introduce two mutant genes into the same cell

2. Grow in restrictive condition

3. Examine effects

-Mutations complement if WT phenotype is restored (mutations in different gene = non-allelic)

-Mutations do not complement if mutant phenotype is observed (mutations in same gene = allelic)

Can only be done on RECESSIVE alleles

Recombination Process

Used to map position of mutations relative to each other within a single gene

-Leads to production of recombinant progeny with phenotypes different than both parents (recombinants are rare)

Mechanism of Recombination

Genetic loci on different DNA molecules are exchanged onto the same DNA molecule

-Forms hybrid DNA molecule different from parental DNA

-Site of breaking/rejoining is called a cross-over

Exchange occurs at random, always reciprocal

Recombination Between Phage Genomes w/ Mutations

1. Phage genomes introduced into same cell by co-infection at high MOI

2. Replicate under permissive conditions:

• Genomes recombine at site or sequence similarity

• Progeny phage produced

3. Progeny are examined under:

• Permissive Conditions - Count total progeny

• Restrictive Conditions - Only WT recombinants (a+, b+) will grow, can determine number of recombinants (wt x 2 = number of recombinants)

Recombination Frequency

Used to map relative position of genes

-RF is proportional to distance between two mutations or genes

Farther apart = better chance of recombination between genes

RF = # of recombinants / # total progeny

Map Unit (m.u.) = RF x 100%