Mutations

  1. Inheritance in Bacteria
    1. The Fluctuation Test of Luria and Delbruck
      1. the prevailing wisdom: bacterial inheritance was believed to be Lamarckian, and different from other organisms
        1. neo-Darwinian: mutations, desirable, undesirable, or neither, occur at random and the desirable ones are passed on to progeny.
        2. Lamarckian or directed-change: bacteria can adapt directly to the environment, and then pass the adaptation on to progeny.
      2. the Fluctuation Test disproved the directed-change hypothesis, and is generally considered to be the beginning of molecular biology
        1. Luria and Delbruck seized upon the different predictions of the two hypotheses: random-mutation hypothesis says that mutations appear prior to the addition of the selective agent, while the directed-change hypothesis says that mutants appear in response to the selective agent.
        2. They used phage T1 as the selective agent.
          1. T1 will kill wild-type E. coli, but mutations in the host receptor protein TonB make the host cell resistant to infection.
          2. If bacteria are spread on agar plate with phage T1, only the resistant mutants will form colonies, and the number of colonies is a measure of the number of mutations
        3. They performed two types of plating
          1. a single large culture grown overnight, and then divided into smaller aliquots and plated with T1
          2. 20 small cultures grown overnight, and then plated with T1
        4. Directed change mechanism would predict no significant difference in the results of the two experiments
        5. But, significant differences were obtained, including the tell-tale "jackpot" mutants found in the small cultures
        6. "fluctuations" in the numbers of resistant mutants in the small culture experiment provided compelling evidence in favor of random-mutation hypothesis.
    2. Lederberg's replica plating analysis provided further supporting evidence
  2. Types of Mutations
    1. endogenous or spontaneous mutations
      1. base substitutions can be transitions or transversions
        1. transitions involve the substitution of a purine for a purine (or a pyrimidine for a pyrimidine) on the same strand
        2. transversions involve the substitution of a purine for a pyrimidine (or vice versa) on the same strand
      2. types of base substitutions
        1. tautomeric shifts and mispairings
        2. deaminations
          1. of cytosine-uracil
          2. of 5 MeC -thymine
          3. hot spots
        3. depurinations can cause transversions
          1. happens when purine is removed during replication
          2. A is often incorporated in new DNA across from AP site
          3. this can lead to transversion in next round of replication
        4. oxidative damage
          1. can form 8 oxo dGuanine
          2. which pairs with A and can also cause transversions
      3. frameshifts and the Streisinger slippage model
        1. role of regions with base repeats
        2. slippage model
        3. phase variation in Bordetella pertussis
          1. bacterium causes whopping cough
          2. virulence genes are switched on and off as a group
          3. mechanism involves a frameshft hotspot
      4. spontaneous and programmed rearrangements
        1. direct repeats can cause deletions by looping out
        2. inverted repeats can cause inversions
    2. induced mutations
      1. base analogs behave as tautomers
      2. nitrous acid induces oxidative deaminations
      3. alkylating agents modify bases, particularly G-OMeG, which pairs with T
      4. hydroxylamine modifies C so that it pairs with A
      5. intercalating agents induce frameshifts
  3. Revertants and suppressors
    1. reversion is the restoration of a mutated function, through the reversal of the original mutation
    2. on the other hand, when one mutation relieves the effect of another mutation, we say that the original mutation has been "suppressed", and the second mutation is called a suppressor.
    3. intragenic (that is, in the same gene) suppressors are very useful, because they often indicate that two amino acids in a protein must interact for the protein to function properly.
    4. intergenic suppressors (that is, the suppressor mutation is in a different gene than the original mutation.
      1. second site suppressors
        1. might occur in one subunit to restore quaternary structure that was disrupted by the first mutation
        2. might have other effects that I'd rather not go into.
      2. nonsense suppressors are mutations, usually  in tRNA genes, that cause the misreading of stop codons
        1. example: the tRNA for glutamine has the anticodon 3'GUC5'.
        2. there are two genes for this tRNA
        3. what if one of the tRNAGln genes is mutated such that the anticodon is 3'AUC5'.  If it is charged normally with glutamine, it will incorporate a glutamine at the UAG stop codons during translation.  We call the strain that carries this mutation a suppressor (specifically, an amber suppressor.  Don't ask why. It doesn't help to know).  Note: the other tRNA gene is normal, so normal glutamine codons can be read correctly by that charged tRNAGln
        4. thus, if another gene has a nonsense mutation that created a UAG stop codon, that mutation will be suppressed if it is placed in the amber suppressor.