1. Ch 8 Microbial Genetics

2. Student Learning Outcomes
Define genetics, genome, chromosome, gene, genetic code, genotype, phenotype, and genomics.
Describe the process of DNA replication.
Describe protein synthesis, including transcription, RNA processing, and translation.
Classify mutations by type, and describe how mutations are prevented and repaired.
Define mutagen.
Describe two ways mutations can be repaired.
Outline methods of direct and indirect selection of mutants.
Identify the purpose and outline the procedure for the Ames test.
Compare the mechanisms of genetic recombination in bacteria.
Differentiate between horizontal and vertical gene transfer.
Describe plasmids and their functions.
© 2004 by Jones and Bartlett Publishers

3. Terminology Genetics Genome Gene Chromosome Base pairs Genetic code
Genomics
Genotype
Phenotype

4. DNA Polymer of nucleotides: ___________________
Double helix associated with proteins
"Backbone" composed of ___________________
Strands are held together by H bonds between ____ and ____
Strands are antiparallel
Fig 8.3b

5. The Bacterial DNA Mostly single circular chromosome
Attached to plasma membrane
DNA is supercoiled
Number of genes in E. coli
Extra-chromosomal bacterial DNA: _________(1-5% of chromosome size)

6. Flow of Genetic Information
Fig 8.2 – Foundation Figure

7. DNA Replication DNA polymerase initiated by RNA primer bidirectional
leading strand: continuous DNA synthesis
lagging strand: discontinuous DNA synthesis  Okazaki fragments
semiconservative 2
Fig 8.3a

8. Replication fork
Replication in 5'  3' direction
Fig 8.5

9. Replication of circular bacterial Chromosome - Three YouTube animations 1; 2; 3
Fig 8.6
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10. Protein Synthesis Transcription Translation
produces 3 types of RNA (?)
Enzyme necessary ?
Promoters and terminators
Translation
produces the protein
Sense codons vs. nonsense codons
anticodons
Genetic code: universal and degenerate (or redundant)
Fig 8.8: Review but do not memorize

11. More Details on Transcription
RNA polymerase binds to promotor sequence
proceeds in 5'  3' direction
stops when it reaches terminator sequence
Fig 8.7
Fig 8.7

12. More Details on Translation
Nucleotide sequence of mRNA is translated into amino acid sequence of protein using “three letter words” = codons
Translation of mRNA begins at the start codon: AUG
Translation ends at a stop codon: UAA, UAG, UGA
Requires various accessory molecules and 3 major components: ?
In Prokaryotes: Simultaneous transcription and translation  Polyribosomes

13. The Translation Process in Protein Synthesis
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Compare to Fig 8.9

14. Simultaneous Transcription and Translation in Prokaryotes
Compare to Fig 8.10

15. Mutations Changes in the genetic material
May be neutral, beneficial, or harmful
Mutagen: Agent that causes mutations
Spontaneous mutations: Occur in the absence of a mutagen
Types of Mutations:
Point mutation = base substitution (silent, missense, nonsense, readthrough)
Frameshift mutation = Insertion or deletion of one or more nucleotide pairs

16. Various Point Mutations
Missense
Nonsense
TAA
Silent

17. What type of mutation? Nonsense mutation Missense mutation
Silent mutation
Point mutation
Frameshift mutation
Review Fig 8.17

18. Mutation Rate and Mutagens
Spontaneous mutation rate = 1 in 109 replicated base pairs or 1 in 106 replicated genes
Mutagens increase mutation rate 10 – 1000x
Chemical mutagens, examples:
Nucleoside (base) analogs have altered base- pairing properties. They can be
randomly incorporated into growing cells (cancer drugs)
only used by viral enzymes (e.g. AZT)
Frameshift mutagens such as intercalating agents (e.g.:, aflatoxin, ethidium bromide)
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19. Fig 8.20 a
Nucleoside Analogs

20. Intercalation
Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication.
Potent carcinogens!

21. Radiation as a Mutagen
Ionizing radiation (x-rays and -rays) causes formation of ions that can react with nucleotides and backbone  leads to deletion mutations (ds breaks)
UV rays lead to thymine dimers (intrastrand bonding)
Photolyases = light repair enzymes (use energy from visible light to fix UV light damage)
Nucleotide excision repair for repair of all mutations

22. Repair Photolyases separate thymine dimers Nucleotide excision repair
Fig 8.21
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23. Mutagen Identification: Ames Test
Wild type vs. mutant
Auxotroph vs. prototroph
Many mutagens are carcinogens
Combine animal liver cell extracts with Salmonella auxotroph
 Expose mixture to test substance
 Examine for signs of mutation in Salmonella, i.e. Look for cells (colonies) that have reverted from his– to his+

24. Ames Reverse Gene Mutation Test
Fig. 8.23

25. Positive or negative Ames test?
Explain what happened

26. Genetic Recombination
Exchange of genes between two DNA molecules
Crossing over occurs when two chromosomes break and rejoin
Fig 8.24

27. Vertical gene transfer: During reproduction between cell generations.
Horizontal gene transfer: Gene transfer between cells of same generation. Leads to genetic recombination.
Three mechanisms of horizontal gene transfer:
Transformation
Conjugation
Transduction
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28. 1) Transformation
“Naked” DNA transfer Recipient cells have to be “competent” Occurs naturally among very few genera (G+ and G–) Simple laboratory treatment will make E. coli competent  workhorse for genetic engineering Griffith’s historical experiment in 1928

29. Griffith’s Experiment to Demonstrate Genetic Transformation
Fig 8.25
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30. Transformation and Recombination
Fig 8.26

31. 2) Conjugation
Plasmid and chromosomal DNA transfer via direct cell to cell contact High efficiency F+ = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka “sex pilus”) Recipient cell (F– ) becomes F+ In some cells F factor integrates into chromosome  Hfr cell R plasmids (R factors) are also transferred via conjugation
Fig 8.27
Review Fig 8.28

32. 3) Transduction
DNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage)
2 types of phage-bacteria interaction:
Generalized transduction happens via lytic cycle caused by virulent phages
Specialized transduction will be covered in Ch 13

33. Generalized Transduction
Fig 8.29
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The End