Lecture Outline:

1. Definitions:

• Haploid- having a single set of chromosomes. In bacteria have a single gene for every trait.
• Gene- a sequence of nucleotides that codes for a functional product.
• Diploid- having two sets of chromosomes (A normal state in eucaryotes). Have two genes encoding for the same trait, not identical but two variations of the same gene (each called an allele).
• Genotype- the sum total of the genetic potential of the cell. Not all genes are expressed at any one time.
• Phenotype- properties being expressed. Phenotype may change with conditions of growth.
• Auxotroph- A laboratory derived mutant that requires an organic growth factor not required by the organism it was derived from, called the prototroph or the wild-type organism.

2. Genetics- the science of heredity. There are several reasons why bacteria have been excellent experimental organisms to study genetics:

• Bacteria are easy to grow.
• They can be grown under a defined set of conditions.
• We can work with a large numbers of like-cells (pure cultures).
• Bacteria are haploid organisms; having a single gene for every trait they possess.
• Mutant cells are fairly easy to screen for provided that the mutation is not lethal.

3. Mutation: any change in the DNA nucleotide sequence. Two general ways that they can occur.

• spontaneously- the spontaneous mutation rate is the probability that a gene will mutate per cell division, and is expressed as a negative exponent. The mutation rate varies with the gene, and mutations occur independent of one another. The spontaneous mutation rate in bacteria can range from 10^-4 to 10^-12 with the average mutation rate being around 10^-6.
• induced- as a result of exposure to what are called mutagenic agents, mutations occur more frequently. Mutagens are not specific to a gene or group of genes, but instead introduce random mutation into the genome.

4. How do spontaneous mutations occur?

• as part of normal DNA replication- DNA replication is not an error free process. What is meant by the proofreading ability of DNA polymerase?
• tautomeric shifts- the nitrogen-containing bases of DNA undergo rare, and reversible, redistribution of electrons and protons in their structure, called tautomeric shifts, which alter their base paring properties. If this occurs at time base is serving as a template will insert wrong base.
• addition/removal of one or more nucleotides as a result of a slippage error during DNA replication (A frameshift mutation).

5. Mutagens:

• chemical mutagens-
  • modifying agents (deaminating agents, remove amino group from a base, and alkylating agents, which add a methyl or ethyl group to a base) change structure of base which alters its base-pairing properties. This is a permeant change, but can be repaired.
  • base analogs- structural analog(s) of a base which upon incorporation into the DNA will base pair differently.
  • intercalating agents- "flat" molecules that insert into the middle of the double helix of DNA, and distort the structure of DNA in that region. This can block DNA replication and/or introduce errors during replication.
• physical agents-
  • ionizing radiation- x-rays, gamma rays.
  • non-ionizing radiation- UV-light causes the formation of thymine dimers (covalent linkage of two adjacent thymine's on same strand of DNA.
• biological mutations or transposable genetic elements- DNA sequences that have ability to replicate themselves independent of DNA they are part of, and to have one copy move, jump, or translocate to another site on that DNA molecule or to another DNA within cell, i.e., a plasmid.
  • insertion sequences (IS elements)- only carry genetic information for transposition.
  • transposons (Tn)- carry additional genetic information such as genes that encode resistance to antibiotics.

6. Types of mutational alterations: Where mutation occurs in a gene will determine effect (functional vs. nonfunctional protein) and/or properties of mutated protein (example, temperature sensitivity).

• silent- genetic code is degenerate, i.e. two different codon's can specific same amino acid.
• missense- one amino acid substituted for another.
• nonsense- a stop codon is generated, which results in a truncated protein.
• frame-shift- the addition/removal of one or two bases will change the reading of the m-RNA. The addition/removal of 3 nucleotides keeps the reading frame intact, but results in a loss/addition of one amino acid.

6. Mutant selection:

• direction selection-
• indirect selection- The Replica Plating technique (isolation of auxotrophic mutants). What are you looking for on replica plates?

5. Repair Mechanisms:

• Proofreading of DNA polymerase
• Light or photoreactivation repiar (Fig. 8.11a)- corrects thymine dimer damage only. Enzyme, photoreactivating enzyme or photolyse, absorbs energy of light, and uses energy to break covalent bond between thymine's.
• Dark or Excision repair (Fig 8.11b). A general repair mechanism. An endonuclease cuts damaged region out, and then DNA polymerase uses undamaged strand as a template to synthesize a new complimentary strand. DNA ligase close gap in DNA.
• Methyl Directed Mismatch repair (Fig. 8.10)
• SOS, Error Prone, Repair

6. Test for mutagens: The Ames Test is an easy and inexpensive way to measure the mutagenicity of a chemical. Can use Ames test to screen for chemicals that have the highest likelihood of being cancinogenic, cancer causing. Rational of test is that greater than 90% of all chemical mutagens have been shown to be carcinogenic in subsequent animal tests. Ames test does not determine if chemical is carcinogenic. How is the test set up, and what is the purpose of the rat liver extract?

Learning Objectives:

• Definition of: a mutation, genotype, phenotype, haploid, auxotroph, mutagen.
• What is the mechanism of action of mutagens?
• What are the consequences of a mutational alteration?
• How can the replica plating technique be used to isolate auxotrophic mutants?
• Describe how DNA damage can be repaired.
• Describe the Ames Test and it's use in identifying carcinogen substances.

 6/18/09