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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 Terminology Complementary but antiparallel Genetics Genome Gene Chromosome Base pairs Genetic code Genomics Genotype Phenotype DNA Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 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) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings E. coli Fig 8.1 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.1a Chromosome Map of E. coli Chromosome length: 1mm Cell length ? Figure 8.1b Flow of Genetic Information Fig 8.2 – Foundation Figure 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 2 Replication fork Replication in 5' 3' direction Fig 5.8 Replication 1; 2; 3 of circular bacterial Chromosome Fig 8.6 Protein Synthesis Genetic code: universal and degenerate (or redundant) Fig 8.8 Transcription produces 3 types of RNA (?) Enzyme necessary ? Promoters and terminators Fig 8.7 Translation produces the protein Sense codons vs. nonsense codons anticodons Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Fig 8.9 Compare to Fig 8.8 Transcription RNA polymerase binds to promotor sequence proceeds in 5' 3' direction stops when it reaches terminator sequence Fig 8.7 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 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 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings The Translation Process in Protein Synthesis Compare to Fig 8.9 Continuous Transcription and Translation Simultaneous Transcription and Translation in Prokaryotes Compare to Fig 8.10 Mutations Change in genetic material. 1. Point mutations = base pair substitution (silent, missense, nonsense) 2. Frameshift mutations = Insertion or deletion of one or more nucleotide pairs Review Fig 8.17 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Various Point Mutations Missense Nonsense Silent Fig 8.17 Mutations cont. 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 basepairing 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) Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Fig 8.19a Distortion due to intercalating agent will lead to one or more base-pairs inserted or deleted during replication. Potent carcinogens! Radiation as a Mutagen 1. Ionizing radiation (x-rays and -rays) lead to deletion mutations (ds breaks) 2. 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 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Repair Photolyases separate thymine dimers Nucleotide excision repair Fig 8.20 ANIMATION Mutations: Repair Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 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+ Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Ames Reverse Gene Mutation Test Fig. 8.22 Professor Richard A. Muller of UC Berkeley on the Ames Test and Natural Foods Positive or negative Ames test? Explain what happened 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 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings 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: 1. Transformation 2. Conjugation 3. Transduction Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings ANIMATION Horizontal Gene Transfer: Overview Genetic Recombination Exchange of genes between two DNA molecules Crossing over occurs when two chromosomes break and rejoin Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Figure 8.23 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 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Griffith’s Experiment to Demonstrate Genetic Transformation Fig 8.24 ANIMATION Transformation Transformation and Recombination Fig 8.25 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+ Fig 8.26 In some cells F factor integrates into chromosome Hfr cell R plasmids (R factors) are also transferred via conjugation Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings ANIMATIONs Fig 8.27 3) Transduction DNA Transfer from donor to recipient cell with help of bacteriophage (= transducing phage) 2 types of phage-bacteria interaction: 1. Generalized transduction happens via lytic cycle caused by virulent phages Fig 8.27 2. Specialized transduction will be covered in Ch 13 Copyright © 2006 Pearson Education, Inc., publishing as Benjamin Cummings Transduction by a Bacteriophage ANIMATION Generalized Transduction ANIMATION Specialized Transduction Fig 8.28