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Download Lecture 9 - Bacterial Genetics Chpt. 8
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2/10/2011 Chapter 8: Bacterial Genetics What are mutations? • Change in the base sequence of the DNA • Do they always change the genetic code? Vertical Gene transfer 1 2/10/2011 Horizontal gene transfer Spontaneous Mutation • Spontaneous mutations occur in natural environment – Occur infrequently and randomly • Rate of mutation – Probability that a mutation will be observed in a given gene each time the cell divides – Rate is generally between 1 in 10,000 and 1 in a trillion • Low rate is due to cellular repair mechanisms Spontaneous Mutation • Mutations are stable heritable changes in the base sequence of DNA • Can occur from – Base substitutions – Removal or addition of nucleotides – Transposable elements 2 2/10/2011 Types of Mutations • Base-pair mutation – Missense mutation – Nonsense mutation – Silent mutation • Frameshift mutation • Transposons Base-pair mutation: missense Results of base-pair mutations 3 2/10/2011 Types of Mutations • Frameshift – Changes the reading frame Frameshift mutation Types of Mutations • Transposable elements – Special segments of DNA that move spontaneously from gene to gene • Elements called transposons – Transposons disrupt proper function of gene • Gene or gene product generally nonfunctional 4 2/10/2011 Barbara McClintock: “jumping genes” biological mutagen What can cause mutations? • Chemicals (nitrous acid) • Physical mutagens (uv light) • Biological mutagens (transposons) Nitrous Acid as a Chemical Mutagen 5 2/10/2011 Nucleoside Analogs are Chemical Mutagens Intercalating Agents as Chemical Mutagens • Molecules that insert themselves between adjacent bases –Create space between bases »Extra base is often added to fill space • Ethidium bromide is common intercalating agent –Potential carcinogen Intercalating Agents 6 2/10/2011 UV Light as a Physical Mutagen • Radiation – Two types • Ultraviolet light – Causes covalent bonding between adjacent thymine bases » Forms thymine dimers which distorts DNA • X-rays – Cause breaks and alterations in DNA » Breaks that occur on both strands are often lethal Repair mechanisms • Repair of base substitution – Cells develop two methods of repair • Proofreading – DNA polymerase has proofreading function – Able to excise incorrect base and replace with correct one • Mismatch repair – Endonuclease enzyme removes short stretch of nucleotide – DNA polymerase fills gap – DNA ligase joins ends 7 2/10/2011 Repair: Mismatch Repair of UV Damage • Repair of thymine dimers – Two mechanisms • Light repair – a.k.a photoreactivation – Enzyme uses visible light to break covalent bonds between bases • Dark repair – a.k.a excision repair – Endonuclease excises damaged section – New section replicated and joined to original strand Light Repair 8 2/10/2011 Dark Repair Repair of Damaged DNA • Repair of modified bases – Enzyme cuts DNA backbone and removes base – DNA polymerase incorporates new base • SOS repair – Last ditch effort to bypass damage – Damage induces SOS system • Produces new DNA polymerase – Highly error prone » Mutations can arise from synthesis with new enzyme Repair of Damaged DNA 9 2/10/2011 Why use bacteria to study mutations? • Only have one chromosome…one copy of each gene • Easy to grow Direct Selection • Testing for traits that are easily identified – Resistance to antibiotics – Motility Direct selection Involves inoculating population of bacteria on medium on which only mutants will grow Used to select antimicrobialresistant organisms 10 2/10/2011 Indirect Selection • A way to look at traits that are not easily identified, e.g. changes in metabolic p pathways y • Replica plating – A way to identify AUXOTROPHS from PROTOTROPHS Indirect selection Required to isolate organisms that require growth factor that parent strain does not Replica plating Testing chemicals for mutagenicity…Ames test 11