chapter 10 prokaryotic genetics. plasmids plasmids: genetic elements that replicate independently of...
TRANSCRIPT
Chapter 10
Prokaryotic Genetics
Plasmids
• Plasmids: genetic elements that replicate independently of the host chromosome– Small circular or linear DNA molecules
– Range in size from 1 kbp to > 1 Mbp; typically less than 5% of the size of the chromosome
– Carry a variety of nonessential, but often very helpful, genes
– Abundance (copy number) is variable
Prokaryotic diversity
• Why are prokaryotes so diverse when they do not reproduce sexually?
• Mutation– Inherited change in genotype– Small gradual change
• Recombination– Integration of DNA (from another organism or
genetic element) into chromosomal DNA– Sometimes very large changes
Mutants
• Some of this we already covered in chapter 6– Often silent
• Change in nucleic acid, but no change in amino acid coded for
– Sometimes phenotypic changes• Requires change in amino acid
• Lethal, neutral, beneficial
– Sometimes a change in amino acid (so not silent) but no change in protein so no phenotypic change
Point mutations
• Involving one base pair• Nucleic acid base substitution
– Missense = changes the codon (1st or 2nd base)wrong amino acid
• Changes protein– Sometimes a phenotypic change and sometimes not
– Nonsense= changes the codon and codes for a stop codon• Translation terminated early protein often non-functional
– Silent= changes last base in codonsame amino acid usually
• Degeneracy of the code
Point mutation: base substitution
Point mutations
• Transitions– One purine base (A or G) is substituted for another
purine or one pyrimidine base (C or T) is substituted for another pyrimidine
• Transversions– A purine is substituted for a pyrimidine or a
pyrimidine is substituted for a purine
Point mutations
• Frameshift mutation– Insertion or deletion of a few nucleotides causing a
reading frame shift and disruption of translation– Insertion= +1 frameshift and deletion = -1
frameshift
Point Mutation: Frameshift
Other Mutations: More Bases
• Large deletions: more likely lethal– Can only be restored by
recombination• Large insertions: often
inactivate gene – Can only be reverted by large
deletion• Translocations: movement
of a large segment from one area to another (ex. Transposons)
• Inversion: orientation of DNA reversed
Wild type versus mutant
• hisC gene codes for HisC protein
• Mutation in the hisC gene are called hisC1, hisC2 etc.
• =genotype
• Phenotype: His + or His –
• His+ - capable of making histidine
• His – not capable of making histidine
Isolation of mutants• Selectable mutants: can select for a phenotype
by subjecting population to a selection factor– Selectable– antibiotic resistance
• Only certain bacteria will grow on a particular antibiotic• We will do a transformation lab where we will grow
bactiera on certain antibiotics• Used for cloning
• Non-selectable – loss of color (may still have a selective advantage in a natural ecosystem, but cannot easily select for the trait in culture)
• Non-selectable mutants have to be screened• Some will have a different color but all will grow
Isolation of mutants
• His C – mutant: cannot make histidine (auxotroph)– Auxotroph: a nutritional mutant (requires a
growth factor that the WT parent did not require)– Prototroph: the WT parent from which the
auxotroph was derived
• Replica plating is one method to screen for nutritional mutants
Replicate plating to isolate auxotrophic mutants: grow with His but not without His
Molecular Basis of Mutation
• Induced mutations
– Those made deliberately
• Spontaneous mutations
– Those that occur without human intervention
– Can result from exposure to natural radiation or oxygen radicals
• Point mutations
– Mutations that change only one base pair
– Can lead to single amino acid change in a protein or no change at all
Types of mutagens
• Chemical– Nucleotide base analogs:
faulty base pairing
Types of mutagens
• Radiation– Nonionizing: causes pyrimidine dimers, which causes
problems with replication and transcription• EX) UV light
– Ionizing: • More energy• Penetrates through glass• Free radicals will damage DNA and disrupt base pairing• X-rays and Gamma radiation
Genetic recombination
• Recombination– Physical exchange of DNA between genetic elements
• Homologous recombination– Process that results in genetic exchange between
homologous DNA from two different sources
A Simplified Version of Homologous Recombination
Genetic exchange in prokaryotes
• Donor DNA is transferred to recipient cell in 3 possible ways– Transformation: free DNA released from one cell
is taken up by another– Transduction: DNA transfer is mediated by a
virus– Conjugation: plasmid transfer with cell to cell
contact
Mechanisms of Transformation in Gram-Positive Bacteria
Transduction
– Transfer of DNA from one cell to another is mediated
by a bacteriophage
• Generalized transduction: DNA derived from virtually
any portion of the host genome is packaged inside the
mature virion
Generalized transduction
Conjugation: Genetic transfer involving cell to cell contact
• Donor cell – contains a conjugative
plasmid – Produces a sex pilus– F plasmid produces F
pilus– Pili make contact with
recipient cell and pull it closer
– Only donor cells produce pili
Transfer of Plasmid DNA by Conjugation
Mobile DNA: Transposable Elements
• Discrete segments of DNA that move as a unit
from one location to another within other
DNA molecules (i.e., transposable elements)
• Transposable elements can be found in all
three domains of life
– First observed by Barbara McClintock