Mutations and Complementation

Wild type

refers to the state of an organism in nature

Mutation

an inheritable change in the DNA sequence of an organism

Mutant

an organism whose genome carries a mutation

Nutritional mutants

• Prototroph: can grow on minimal medium

• Auxotroph: requires nutritional supplements in order to grow on minimal medium

Mutagen

an agent that induces mutations

Classes of Mutations

• Spontaneous Mutations

• Induced Mutations

Spontaneous Mutations

result from errors during DNA replication

Induced Mutations

mutations caused by exposure to mutagens

Point Mutations

a single base change

• Transition

• Transversion

• Framshift

Transition Mutation

A=T mutates to A=C or G=C mutates to G=T

• common & often repaired

• point mutation

Transversion Mutation

purine substituted w/ pyrimidine (ex: G substitutes w/ T or C)

• rare

• point mutation

Frameshift Mutation

base is added or subtracted → changes reading frame

• point mutation

Phenotypes of Point Mutations

• Silent Mutation

• Missense Mutation

• Nonsense Mutation

Silent Mutation

no phenotypic change

• still codes for the same amino acid sequence

Missense Mutation

results in an amino acid change (may be conditional mutations)

• phenotype = context dependent

Nonsense Mutation

results in the insertion of a stop codon in place of an amino acid

Insertion Mutation

a genetic mutation where one or more nucleotides are added into a DNA sequence, potentially altering the protein produced

Polar Mutation

an insertion mutation that affects not only the gene where it occurs but also reduces or stops expression of downstream genes in the same operon

Types of Mutagens

• Base analogs

• Base-modifying chemicals

• Intercalating agents

Base analogs

chemicals w/ structural similarity to normal nitrogenous bases that base pair differently

• ex: 5-bromouracil (structural analog of T) - acts more like C so base pairs w/ G

Base-modifying chemicals

change base so it base pairs incorrectly

• ex: methyl-nitrosoguanidine: methylates G so that it base pairs with T

Intercalating agents

fit between base pair stacks of DNA & causes single base insertions & deletions

• ex: ethidium bromide

Selection of Mutants

process of isolating and identifying organisms with specific mutations by using conditions that favor their growth over non-mutants

• Direct Selection

• Indirect Selection

Direct Selection of Mutants

look for mutants that are resistant to a particular toxic compound

Indirect Selection of Mutants

look for mutants by replica plating

• commonly used to find nutritional mutants

Complementation

the ability of one set of genes in a merodiploid cell to compensate for a mutation in another set of genes

Testing for Complementation - Transformation

• competent bacteria + plasmid

• select for plasmid encoded phenotype (usually antibiotic resistance)

• select or screen for desired phenotype

Testing for Complementation - Conjugation

• mix donor & recipient bacteria

• select for donor plasmid phenotype & recipient cell phenotype while counter-selecting against donor cell phenotype

• select or screen for the desired phenotype