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

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

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

E. coli Fig 8.1 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 Complementary and antiparallel 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) 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

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

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

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

Various Point Mutations Missense Nonsense Silent

Fig 8.17

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)

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 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

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

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.  

Ames Reverse Gene Mutation Test Fig. 8.22

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

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

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

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

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+ In some cells F factor integrates into chromosome  Hfr cell R plasmids (R factors) are also transferred via conjugation Fig 8.26

Fig 8.27 ANIMATIONs

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

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