Chapter 10

1. Chapter 10 Prokaryotic Genetics

2. 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

4. 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

5. 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

6. 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 codonsame amino acid usually • Degeneracy of the code

7. Point mutation: base substitution

8. 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

9. 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

10. Point Mutation: Frameshift

11. 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 Other Mutations: More Bases

12. 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

13. 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

14. 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

15. Replicate plating to isolate auxotrophic mutants: grow with His but not without His

16. 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

17. Types of mutagens • Chemical • Nucleotide base analogs: faulty base pairing

18. 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

20. 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

21. A Simplified Version of Homologous Recombination

22. 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

23. Mechanisms of Transformation in Gram-Positive Bacteria

24. 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

25. Generalized transduction

26. 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

27. Transfer of Plasmid DNA by Conjugation

28. 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