Bact 303Bacterial Genetics II

Mutation

Mutation is a change in the sequence of bases in DNA

If a gene that encodes for a specific protein is mutated, it may result in a change in the sequence of amino acids comprising the protein. The activity of the protein may be altered.

Frequency of naturally occurring (spontaneous) mutation varies from 10-6 to 10-9 (avg = 10-8)

This means that if a bacterial population increases from 108 to 2 x 108, on the average, one mutant will be produced for the gene in question.

Classes of Mutations

Mutations result from from two underlying causes

1. substitutions - One base is substituted in the DNA for another base. Incorrect base pairing results from the change of a single nucleotide base. Substitutions usually result in “point mutations”

2. insertions or deletions of one or more bases - may result in the the addition or deletion of one or more amino acids to the growing protein, or more likely result in a “frame-shift” mutation

A segment of the genetic code

DNA mRNA codon tRNA anticodon Amino acid

ACG UGC ACG CYSTEINECCT GGA CCU GLYCINEAAA UUU AAA PHENYLALANINECGA GCU CGA ALANINETCG AGC UCG SERINECCG GGC CCG GLYCINETAA AUU UAA ISOLEUCINEACT UGA None None

A C G C C T A A A C G A DNA Transcription

U G C G G A U U U G C U mRNA Translation

_____ _____ _____ _____ cys gly phe ala Protein

A C G C C T A A A C G A DNA

U G C G G A U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

TEMPLATE

A C G C C T A A A C G A DNA

U G C G G A U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

A C G C C T A A A C G A DNA

U G C G G A U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

T C G C C T A A A C G A DNA

A G C G G A U U U G C U mRNA_____ _____ _____ _____ ser gly phe ala Protein

Substitution mutation T--->A at position #1 in DNA changes the mRNA codon to code for the amino acid serine in place of cysteine. This is a missense mutation

A C G C C T A A A C G A DNA

U G C G G A U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

Substitution mutation in the third base of DNA G--->Tgenerates terminator “stop” codon which terminates the translation of the protein. A shortened or “truncated” protein may be produced. This is called a nonsense mutation.

A C T C C T A A A C G A DNA

U G A G G A U U U G C U mRNA_____ -------------------------> stop no translation

A C G C C T A A A C G A DNA

U G C G G A U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

Substitution mutation in the sixth base of DNA T--->Ccauses degenerate codon for glycine so there is no change in the primary structure of the protein. This is called a silent mutation.

A C G C C C A A A C G A DNA

U G C G G G U U U G C U mRNA_____ _____ _____ _____ cys gly phe ala Protein

Consequences of substitution mutations

• none - altered protein retains same enzymatic or structural activity

• change in enzymatic activity - could be beneficial or harmful to the organism

• termination - incomplete (truncated) protein produced

A C G C C G T A A A C G A DNA <---- insert into DNA

U G C G G C A U U U G C U mRNA_____ _______ _____ _____ <---- old reading frame

cys gly phe ala Protein

Mutation caused by the insertion of a new base (G) between bases 5 and 6 in the DNA (gene). When 1 or 2 bases are inserted, this causes a “frame shift” mutation because the translation reading frame becomes shifted by one or two bases generating a whole new set of triplets beyond the mutation. The same is true for mutations that result in the deletion of bases.

A C G C C G T A A A C G A DNA

U G C G G C A U U U G C U mRNA_____ _____ _____ _____ <---- new reading frame

cys gly isoleu cys Protein

Consequences of a frame shift mutation

• usually a more severe type of mutation that will lead to translation of a totally different protein or it will generate a stop codon that will terminate protein synthesis.

Mutagenic agents or mutagens

• Mutagens are physical, chemical or biological agents that increase the rate of spontaneous mutation.

• Some mutagens cause point mutations and some cause frame-shift mutation and some cause both.

Examples of chemical mutagens

CHEMICAL ACTION

HNO2 React chemically with one or more bases so

Nitrogen mustard that they pair improperly

NTG

Intercalating agents Insert into DNA and cause frame-shift (acridine dyes) mutations by inducing an addition or the

subtraction of a base

Base analogs Incorporate into DNA and cause mispairing

e.g.

5-bromouracil Analog of T which can pair with C

2-amino purine Analog of A which can pair with C

Examples of physical mutagens

PHYSICAL AGENT ACTION

UV irradiation Causes formation of adjacent T-T dimers that distorts the DNA backbone, altering the binding properties of bases near the dimer

X-ray Alters bases chemically, causes deletions and induces breaks in DNA chain

Examples of biological mutagens

BIOLOGICAL AGENT ACTION

Insertion sequences (IS) Pieces of DNA about a thousand nucleotide bases in length which can insert into a genetic sequence

Transposons genetic elements goverened by IS which can insert into the chromosome within a gene

Viruses Some bacteriophage (e.g. phage mu) can integrate their DNA into random positions in the bacterial chromosome

Designations for Bacterial MutantsCLASS DESIGNATION EXAMPLE

NUTRITIONAL MUTANTS1. Lack ability to utilize a carbon source Sub- Lac-

2. Have ability to utilize a carbon source Sub+ Lac+

STRUCTURAL MUTANTS1. Lack fimbriae Fim- Fim-

2. Lost motility Mot- Mot-

3. Lost flagella Fla- Fla-

RESISTANCE TO ANTIBIOTIC ABr Ampr

SENSITIVITY TO ANTIBIOTIC ABs Amps

AUXOTROPHIC MUTANT1. Blocked in metabolic pathway for synthesis of an amino acid AA- Trp-

Wild type AA+ Trp+

2. Blocked in metabolic pathwayfor the synthesis of a vitamin Vit- Thi-

Wild type Vit+ Thi+

Detection of Mutations in Bacteria

DIRECT SELECTION OR ENRICHMENT (for Selective Mutants)

1. Plate the bacteria on a medium on which a mutant but not the parent will grow.

2. Antibiotic or virus senstive parents are killed or do not grow. Dru resistant or virus resistant mutants will grow.

3. Pick and purify colony. Confirm resistance.

Direct Selection of Antibiotic Resistance Mutant

Direct Selection of mutants able to use a specific carbon source

Isolation of a motility mutant by direct selection

Isolation of an auxotrophic revertant by direct selection

The Ames Test

Modification of the Ames Test

Detection of Mutations in Bacteria

PENICILLIN SELECTION OR ENRICHMENT (for Nutritional or Auxotrophic Mutants - mutants that require a growth factor not needed by the parent)

1. Add penicillin to culture growing in a medium without the growth factor: a. growing bacteria (can make their own growth factor) are killed in the presence of penicillinb. nongrowing bacteria (because they require the missing growth factor) are not killed (Note: penicillin kills ONLY growing cells)

2. Remove Penicillin, add growth factor and auxotrophs (GF-) will grow. This step selects or enriches for the growth of the survivors.

3. Select a survivor colony, and pick, purify and confirm growth factor requirement (auxotrophy).

Detection of Mutations in Bacteria

REPLICATE PLATING (Also for Nutritional or Auxotrophic Mutants)

1. Transfer part of every colony growing on a complete medium (contains growth factor) to a minimal medium (lacks the growth factor).

2. Identify colonies growing on the complete medium which do not grow on the minimal medium. These are auxotrophic colonies - they grow with, but not without, the added growth factor.

3. Pick and purify auxotrophic colonies from the complete medium and confirm auxotrophy.

Replicate Plating Technique