Biology – 261

Chapter 10
- Mutation: heritable change in DNA sequence that can lead to a change in phenotype
(observable properties of an organism)
- Mutant: A strain of any cell or virus differing from parental strain in genotype (nucleotide
sequence of genome)
- Wild-type strain: typically refers to strain isolated from nature
- Mutations and Mutants
o Selectable mutations: you can select (anything that grows on amp media is resistant to
ampicillin
 Those that give the mutant a growth advantage under certain environmental
conditions
 Useful in genetic research
o Nonselectable mutations: ones that just happen and are more difficult to find (pigment
change)
 Those that usually have neither an advantage nor a disadvantage over the
parent
 Detection of such mutations requires examining a large number of colonies and
looking for differences (screening)
 Screening: process of looking for the colonies that have a specific color od
phenotype that is nonsense
o Screening is always more tedious than selection
 Look at hundreds of colonies to find phenotypes that you want where as
selection whatever grows on selective media is what you are looking for
 Methods available to facilitate screening
 Replica plating  looks for mutant (auxotroph)
 Replica plating is useful for identification of cells with a nutritional requirement
for growth
- Molecular basis of mutation
o Induced mutations: intentionally exposed a cell to a mutagen and as a result of the
exposure a mutation occurs
 Those made deliberate
 Can result from exposure to natural radiation or oxygen or oxygen radicals
o Spontaneous mutations (just happens)
 Those that occur without human intervention
 Can result from exposure to natural radiation or oxygen radicals
o Point mutations (when only one base changes either through deletion, substitution, or
addition, the mutation is called a point mutation)
 Mutations that change only one base pair (add or take out)
 Can lead to a single amino acid change in a protein or no change at all
o Note: one base can change the reading frame cause a change in the protein coded for
by the gene
o Base – substitution mutations
 Silent mutations: does not affect amino acid sequence
 Effect genotype but not phenotype
 Missense mutation: amino acid changed; polypeptide altered
 Creates different codon
 Effects genotype and phenotype
 Nonsense mutation: codon becomes stop codon because it does not code for
amino acid; polypeptide is incomplete
 Note: sense codons code sense amino acids
o Deletions and insertions cause more dramatic changes in DNA
 Deletions: bases removed from DNA
 Insertions: bases are added to DNA
o Frameshift mutations
  Deletions or insertions that result in a shift in the reading frame that may or may
not change amino acid shape, sequence, and function of protein  change in
every codon and amino acid
o Genetic engineering allows for the introduction of specific mutations (site-directed
mutagenesis)
o Point mutations are typically reversible
o Reversion: Alteration in DNA that reverse the effects of a prior mutation
 Single point mutation can be reversed/ corrected
o Revertant: cells which undergo reversions
 Strains in which phenotype that was changed in the mutant is restored
 Two typed
 Same-site Revertant: mutation restoration activity is at the same site as
the original mutation
 Second site Revertant: mutation is at a different site in the DNA
o Suppressor mutation that compensates for the effect of the original
mutations
- Mutation rates: vary with mutation and organisms but generate very low due to proof reading
capabilities of poly
o For most microorganisms, errors in ENS replication occur at a frequency of 10 to the
neg 6 to 10 to the neg 7 per kilobase
o DNA viruses have error rates 100 – 1,000 X greater
o The mutation rate in RNA genomes in 1,000 – fold higher than in DNA genomes
 Some RNA poly have proofreading capabilities
 Comparable RNA repair mechanisms do not exist
- Mutagenesis: chemical, physical, or biological agents that increase mutation rates (whatever
causes mutation)
o Several classes of chemical mutagens exist
 Nucleotide base analogs: resemble nucleotides
 Chemical mutagens can induce chemical modifications
 Acridines: intercalating agents; typically cause Frameshift mutations
o Several forms of radiation are highly mutagenic
o Two main categories of mutagenic electromagnetic radiation
 Non-ionizing (UV radiation):
 Causes T-T dimes to form in cell which if not repaired can lead to skin
cancer
 Purines and pyrimidines strongly absorb UV
 Pyrimidine dimers is one effect of UV radiation
 Ionizing (X-rays, cosmic rays, gamma rays):
 All the way through the skin, breaks the DNA which can lead to death
 Ionize water and produce free radicals
 Free radicals damage macromolecules in the cell
 Dosage is important
 More radiation, more mutations, more death or malignant cells
o When DNA damage is large scale the cell may need to use a different type of repair
system (damage interferes with DNA replication)
 This system is more error prone
 Allows replication to proceed and cell to replicate, but errors are more likely
 Mechanism called the SOS regulatory system (main repair mechanism studied in
bacteria)
 Translesion synthesis allows DNA to be synthesized with no template
o Perfect fidelity in organisms is counterproductive because it prevents evolution
o The mutation rate of an organism is subject to change
 Mutants can be isolated that are hyper accurate or have increased mutation
rates
 Mutations = genetic diversity
  Spontaneous mutation rate = low/ relatively stable
- Mutagenesis and Carcinogenesis: The Ames Test
o The Ames test (used in food industry) makes practical use of bacterial mutation to
detect for potentially hazardous chemicals
 Looks for an increase in mutation of bacteria in the presence of suspected
mutagen
 A wide variety of chemicals have been screened for determining carcinogenicity
- Transformation: uptake of free (plasmid) DNA
o Genetic transfer process by which DNA is incorporated into a recipient cell and brings
about genetic change
o Discovered by Fredrick Griffith in the late 1920s
 Worked with Streptococcus pneumococcus
o This process set the stage for the discovery of DNA
o When cells die they sometimes lyse and release plasmid, if new bacteria are nearby
they can pick up that plasmid
o Competent: cells capable of taking up DNA and being transformed (when a cell can
undergo transformation)
 Must be competent to go through transformation
 In naturally transformable bacteria, competence is regulated
 In other strains, specific procedures are necessary to make cells competent and
electricity can be used to force cells to take up DNA (electroporation)
 Bacteria are not competent
o Curing natural transformation, integration of transforming DNA is a highly regulated,
multi-step process
o Transfection (similar to transformation except DNA comes from a phage rather than
another bacterium)
 Transformation of bacterium with DNA extracted from a bacterial virus
- Transduction
o Transfer of DNA from one cell to another is mediated by a bacteriophage
o Two methods
 Generalized: DNA derived from virtually any portion of the host genome is
packaged inside the mature virion
 Defective virus particle incorporates fragments of the cell’s chromo
randomly
 Can be temperate or virulent
 Low efficient
 Transferring bacterial DNA from one to another via the lytic phase of a
bacteriophage when transducing phage infects new cell genes aren’t
transferred but new cell doesn’t get any phage DNA and thus doesn’t lyse
 Normal virions contain phage gene while transducing particle contain host
genes
 Specialized: DNA from a specific region of the host chromo is integrated directly
in the virus genome
 DNA of temperate virus excises incorrectly and takes adjacent host genes
along with it
 Transducing efficiency can be high
- Conjugation: Essential features
o Bacterial conjugation (mating): mechanism of genetic transfer that involves cell-to-cell
contact
 Plasmid encoded mechanism
 Donor cell: contains conjugative plasmid (F+)
 Recipient cell: does not contain plasmid (F-)
 Cells must touch one another
 Pili how cells connect
 F+ and F- cells connect
  F+ cells give F factor to F- cell making it F+
 F factor = plasmid (ability to making pilus)
o F (fertility) plasmid: circular DNA molecule ~ 100 kbp
o Contains genes that regulate DNA replication
o Contain several transposable elements that allow the plasmid to integrate into the host
chromo
o Contains tra gene that encode transfer functions
o Transferred from F= to F-  single copy
o HFR conjugation (high frequency)  does not give complete copy of F factor to F –
o Sex pilus is essential for conjugation
 Only produced by donor cell
o Conjugation occurs when a cell has the F plasmid and is able to make a pilus
o DNA synthesis is necessary for DNA transfer by conjugation
 DNA synthesis by rolling circle replication (mechanism of transfer of the F
plasmid)
 Also used by some viruses
- Formation of Hfr strains and chromo mobilization
o F plasmid is an episome; can integrate into host chromo  cells called hfr cells
o Cells possessing a non-integrated F plasmid are cell F+
o Cells possessing an integrated F plasmid are called hfr (high frequency recombination)
 High rates of genetic recombination between genes on the donor chromo and
those of the recipient
o Presence of the F plasmid results in alterations in cell properties
 Ability to synthesis F pilus
 Mobilization of DNA for transfer to another cell
 Alteration of surface receptors so that cell can no longer act as a recipient in
conjugation
o Cells have F plasmids = make pilus, transfer plasmids to new cell in which alters
recipient cell to receive the plasmid and in some cases chromo gene from donor cell
o Insertion sequence (mobile elements) are present in both the F plasmids and E. coli
chromo
 Facilitate homo recombination
o Plasmid is now part of chromo
 Chromo genes transferred with plasmid
o Recipient cell does not become hfr because only a portion of the integrated F plasmid
is transferred by the donor
- Mobile DNA: Transposable Elements
o Discrete segments of DNA that move as a unit from one location to another within
other DNA molecules (transposable elements)
o Transposable elements can be found in all three domains of life
o Move by a process called transportation
 Frequency of transportation is 1 in 1,000 to 1 in 10,000,000 per generation
 First observed by Barbara McClintock
o Two main types of transposable elements in Bacteria are transposons and insertion
sequences
 Insertion sequene are the simplest transposable element
 ~1,000 nucleotides long
 Inverted repeats are 10-50 base pairs
 Only gene is for the transposase
 Found in plasmids and chromo of bacteria and archaea and some
bacteriophage
 Smaller than trans
 Transposons
 Larger than insertion sequences
 Transposase moves any DNA between inverted repeats and cuts DNA
  May include antibiotic resistance
 Examples are the tn5 and the tn10
 Carry with them other genes like genes for antibiotic resistance
 Both carry genes encoding transposase
 Both have inverted repeats at their ends
o Transposons and insertion sequences are able to move around on the chromo which
creates mutations