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  • 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
  • Extra-chromosomal (mostly circular) DNA - replicates independently of chromosome
  • Complementary and antiparallel
  • 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.  
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    1. 1. 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 Objectives
    2. 2. Terminology <ul><li>Genetics </li></ul><ul><li>Genome </li></ul><ul><li>Gene </li></ul><ul><li>Chromosome </li></ul><ul><li>Base pairs </li></ul><ul><li>Genetic code </li></ul><ul><li>Genomics </li></ul><ul><li>Genotype </li></ul><ul><li>Phenotype </li></ul>Complementary but antiparallel DNA
    3. 3. The Bacterial DNA <ul><li>Mostly single circular chromosome </li></ul><ul><li>Attached to plasma membrane </li></ul><ul><li>DNA is supercoiled </li></ul><ul><li>Number of genes in E. coli </li></ul><ul><li>Extra-chromosomal bacterial DNA: _________ (1-5% of chromosome size) </li></ul>
    4. 4. E. coli Figure 8.1a Fig 8.1
    5. 5. Figure 8.1b Chromosome Map of E. coli Chromosome length:  1mm Cell length ?
    6. 6. Flow of Genetic Information Fig 8.2 – Foundation Figure
    7. 7. DNA Replication <ul><li>DNA polymerase initiated by RNA primer </li></ul><ul><li>bidirectional </li></ul><ul><li>origin of replication </li></ul><ul><li>leading strand: continuous DNA synthesis </li></ul><ul><li>lagging strand : discontinuous DNA synthesis  Okazaki fragments </li></ul><ul><li>semiconservative </li></ul>2
    8. 8. Replication fork <ul><ul><li>Replication in 5'  3' direction </li></ul></ul>Fig 5.8
    9. 9. Fig 8.6 Replication 1 ; 2 ; 3 of circular bacterial Chromosome
    10. 10. Protein Synthesis <ul><li>Genetic code: universal and degenerate (or redundant) </li></ul><ul><li>Transcription </li></ul><ul><ul><li>produces 3 types of RNA (?) </li></ul></ul><ul><ul><li>Enzyme necessary ? </li></ul></ul><ul><ul><li>Promoters and terminators </li></ul></ul><ul><li>Translation </li></ul><ul><ul><li>produces the protein </li></ul></ul><ul><ul><li>Sense codons vs. nonsense codons </li></ul></ul><ul><ul><li>anticodons </li></ul></ul>Fig 8.9 Fig 8.7 Fig 8.8
    11. 11. Compare to Fig 8.8
    12. 12. Transcription <ul><li>RNA polymerase binds to promotor sequence </li></ul><ul><li>proceeds in 5'  3' direction </li></ul><ul><li>stops when it reaches terminator sequence </li></ul>Fig 8.7
    13. 13. More Details on Translation <ul><li>Nucleotide sequence of mRNA is translated into amino acid sequence of protein using “three letter words” = codons </li></ul><ul><li>Translation of mRNA begins at the start codon: AUG </li></ul><ul><li>Translation ends at a stop codon: UAA, UAG, UGA </li></ul><ul><li>Requires various accessory molecules and 3 major components: ? </li></ul><ul><li>In Prokaryotes : Simultaneous transcription and translation  Polyribosomes </li></ul>
    14. 14. The Translation Process in Protein Synthesis Compare to Fig 8.9
    15. 15. Continuous Transcription and Translation Simultaneous Transcription and Translation in Prokaryotes Compare to Fig 8.10
    16. 16. Mutations <ul><li>Change in genetic material. </li></ul><ul><li>Point mutations = base pair substitution (silent, missense, nonsense) </li></ul><ul><li>Frameshift mutations = Insertion or deletion of one or more nucleotide pairs </li></ul>Review Fig 8.17
    17. 17. Various Point Mutations Silent Missense Nonsense
    18. 18. Fig 8.17
    19. 19. Mutations cont. <ul><li>May be neutral (silent), beneficial, or harmful. </li></ul><ul><li>Spontaneous mutation rate  10 -6  1 mutation per million replicated genes </li></ul><ul><li>Mutagens increase mutation rate 10 – 1000x </li></ul><ul><li>Chemical mutagens </li></ul><ul><ul><li>Nucleoside (base) analogs have altered base-pairing properties. They can be </li></ul></ul><ul><ul><ul><li>randomly incorporated into growing cells (cancer drugs) </li></ul></ul></ul><ul><ul><ul><li>only used by viral enzymes (e.g. AZT) </li></ul></ul></ul><ul><ul><li>Frameshift mutagens such as intercalating agents ( e.g.: , aflatoxin, ethidium bromide) </li></ul></ul>
    20. 20. Fig 8.19a
    21. 21. Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication. Potent carcinogens!
    22. 22. Radiation as a Mutagen <ul><ul><li>Ionizing radiation (x-rays and  -rays) lead to deletion mutations (ds breaks) </li></ul></ul><ul><ul><li>UV rays lead to thymine dimers (intrastrand bonding) </li></ul></ul><ul><ul><li>Photolyases = light repair enzymes (use energy from visible light to fix UV light damage) </li></ul></ul><ul><ul><li>Nucleotide excision repair for repair of all mutations </li></ul></ul>
    23. 23. Repair <ul><li>Photolyases separate thymine dimers </li></ul><ul><li>Nucleotide excision repair </li></ul>Fig 8.20 ANIMATION Mutations: Repair
    24. 24. Mutagen Identification: Ames Test <ul><li>Wild type vs. mutant </li></ul><ul><li>Auxotroph vs. prototroph </li></ul><ul><li>Many mutagens are carcinogens </li></ul><ul><li>Combine animal liver cell extracts with Salmonella auxotroph </li></ul><ul><li> Expose mixture to test substance </li></ul><ul><li> Examine for signs of mutation in Salmonella, i.e. Look for cells (colonies) that have reverted from his – to his + </li></ul>
    25. 25. Fig. 8.22 Ames Reverse Gene Mutation Test
    26. 26. Positive or negative Ames test? Explain what happened Professor Richard A. Muller of UC Berkeley on the Ames Test and Natural Foods
    27. 27. Genetic Transfer and Recombination <ul><li>Vertical gene transfer: Occurs during reproduction between generations of cells. </li></ul><ul><li>Horizontal (lateral) gene transfer: Transfer of genes between cells of the same generation. Leads to genetic recombination </li></ul><ul><li>Three mechanisms of horizontal gene transfer: </li></ul><ul><ul><li>Transformation </li></ul></ul><ul><ul><li>Conjugation </li></ul></ul><ul><ul><li>Transduction </li></ul></ul>
    28. 28. <ul><li>Vertical gene transfer : Occurs during reproduction between generations of cells. </li></ul><ul><li>Horizontal gene transfer : The transfer of genes between cells of the same generation. Leads to genetic recombination. </li></ul><ul><li>Three mechanisms of horizontal gene transfer: </li></ul><ul><ul><ul><li>Transformation </li></ul></ul></ul><ul><ul><ul><li>Conjugation </li></ul></ul></ul><ul><ul><ul><li>Transduction </li></ul></ul></ul>Genetic Recombination ANIMATION Horizontal Gene Transfer: Overview
    29. 29. Genetic Recombination <ul><li>Exchange of genes between two DNA molecules </li></ul><ul><li>Crossing over occurs when two chromosomes break and rejoin </li></ul>Figure 8.23
    30. 30. 1) Transformation <ul><li>“ Naked” DNA transfer </li></ul><ul><li>Recipient cells have to be “competent” </li></ul><ul><li>Occurs naturally among very few genera (G+ and G – ) </li></ul><ul><li>Simple laboratory treatment will make E. coli competent  workhorse for genetic engineering </li></ul><ul><li>Griffith’s historical experiment in 1928 </li></ul>
    31. 31. Griffith’s Experiment to Demonstrate Genetic Transformation ANIMATION Transformation Fig 8.24
    32. 32. Transformation and Recombination Fig 8.25
    33. 33. 2) Conjugation <ul><li>Plasmid and chromosomal DNA transfer via direct cell to cell contact </li></ul><ul><li>High efficiency </li></ul><ul><li>F + = donor cell. Contains F plasmid (factor) and produces conjugation (F) pilus (aka “sex pilus”) </li></ul><ul><li>Recipient cell (F – ) becomes F + </li></ul><ul><li>In some cells F factor integrates into chromosome  Hfr cell </li></ul><ul><li>R plasmids (R factors) are also transferred via conjugation </li></ul>Fig 8.26
    34. 34. Fig 8.27 ANIMATIONs
    35. 35. 3) Transduction <ul><li>DNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage) </li></ul><ul><li>2 types of phage-bacteria interaction: </li></ul><ul><ul><li>Generalized transduction happens via lytic cycle caused by virulent phages </li></ul></ul><ul><ul><li>Specialized transduction will be covered in Ch 13 </li></ul></ul>Fig 8.27
    36. 36. Transduction by a Bacteriophage ANIMATION Generalized Transduction ANIMATION Specialized Transduction Fig 8.28 The End