Chapter 10 Prokaryotic Genetics. Plasmids Plasmids: genetic elements that replicate independently of...

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