1. Describe the process of DNA replication.
Objectives
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 the functions of plasmids and transposons.
© 2004 by Jones and Bartlett Publishers

2. Terminology DNA Genetics Genome Gene Chromosome Base pairs
Genetic code
Genomics
Genotype
Phenotype
Complementary but antiparallel
DNA
Genetics: The study of what genes are, how they carry information, how information is expressed, and how genes are replicated.
Gene: A segment of DNA that encodes a functional product, usually a protein.
Genome: All of the genetic material in a cell
Genomics: The molecular study of genomes
Genotype: The genes of an organism
Phenotype: Expression of the genes

3. 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)
Extra-chromosomal (mostly circular) DNA - replicates independently of chromosome

4. E. coli
Fig 8.1
Figure 8.1a

5. Chromosome Map of E. coli
Chromosome length:  1mm
Cell length ?
Figure 8.1b

6. Flow of Genetic Information
Fig 8.2 – Foundation Figure

7. DNA Replication DNA polymerase initiated by RNA primer bidirectional
origin of replication
leading strand: continuous DNA synthesis
lagging strand: discontinuous DNA synthesis  Okazaki fragments
semiconservative
Complementary and antiparallel 2

8. Replication fork
Replication in 5'  3' direction
Fig 5.8

9. Replication 1; 2; 3 of circular bacterial Chromosome
Fig 8.6

10. Protein Synthesis
Genetic code: universal and degenerate (or redundant)
Transcription
produces 3 types of RNA (?)
Enzyme necessary ?
Promoters and terminators
Translation
produces the protein
Sense codons vs. nonsense codons
anticodons
Fig 8.8
Fig 8.7
Fig 8.9

11. Compare to Fig 8.8

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

13. 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

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

15. Continuous Transcription and Translation
Simultaneous Transcription and Translation in Prokaryotes
Compare to Fig 8.10

16. Mutations Change in genetic material.
Point mutations = base pair substitution (silent, missense, nonsense)
Frameshift mutations = Insertion or deletion of one or more nucleotide pairs
Review Fig 8.17

17. Various Point Mutations
Missense
Nonsense
Silent

18. Fig 8.17

19. Mutations cont. Chemical mutagens
May be neutral (silent), beneficial, or harmful.
Spontaneous mutation rate  10-6  1 mutation per million replicated genes
Mutagens increase mutation rate 10 – 1000x
Chemical mutagens
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)

20. Fig 8.19a

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

22. Radiation as a Mutagen
Ionizing radiation (x-rays and -rays) lead 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

23. Repair Photolyases separate thymine dimers Nucleotide excision repair
Fig 8.20
ANIMATION Mutations: Repair

24. 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+
Strong correlation between mutagen strength and carcinogen strength in rodent studies.
Um zu prüfen, ob eine Chemikalie mutagen wirkt führt man den Ames-Test durch. Er beruht auf der Annahme, daß jeder Stoff, der als Mutagen wirkt auch karzinogen ist.
Links ist eine Mutante des Bakteriums Salmonella typhimurium zu sehen, das kein Histidin (His) synthetisieren kann. Der Agar enthält Rattenleber-Enzyme und kein Histidin. Der Papierfilter wurde mit 10µg des Karzinogens 2-Aminofluorin getränkt. Die krebserregende Wirkung sorgte für bei vielen Bakterien für Rückmutationen, denn sie wachsen trotz Histidin-Abwesenheit.

25. Ames Reverse Gene Mutation Test
Fig. 8.22

26. Positive or negative Ames test?
Professor Richard A. Muller of UC Berkeley on the Ames Test and Natural Foods
Positive or negative Ames test?
Explain what happened

27. Genetic Transfer and Recombination
Vertical gene transfer: Occurs during reproduction between generations of cells.
Horizontal (lateral) gene transfer: Transfer of genes between cells of the same generation. Leads to genetic recombination
Three mechanisms of horizontal gene transfer:
Transformation
Conjugation
Transduction

28. Genetic Recombination
Vertical gene transfer: Occurs during reproduction between generations of cells.
Horizontal gene transfer: The transfer of genes between cells of the same generation. Leads to genetic recombination.
Three mechanisms of horizontal gene transfer:
Transformation
Conjugation
Transduction
ANIMATION Horizontal Gene Transfer: Overview

29. Genetic Recombination
Exchange of genes between two DNA molecules
Crossing over occurs when two chromosomes break and rejoin
Figure 8.23

30. 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

31. Griffith’s Experiment to Demonstrate Genetic Transformation
Fig 8.24
ANIMATION Transformation

32. Transformation and Recombination
Fig 8.25

33. 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 8.26

34. Fig 8.27
ANIMATIONs

35. 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
Fig 8.27

36. Transduction by a Bacteriophage
ANIMATION Generalized Transduction
ANIMATION Specialized Transduction
The End
Fig 8.28