Lecture 211)Compare and contrast selectable and non-selectable mutations a) Selectablei) Confer a clear advantage to the organism in certain conditionsb) Non- selectablei) Do not confer a particular advantage to the organism (color loss in a pigmented organism)2)Compare and contrast the three types of point mutations a) Missensei) Get faulty protein, might affect the protein function or might not, depends on location and type of mutationb) Nonsensei) Typically get stop codon and stops translation and you can lose protein activity c) Silenti) Does not change the proteinii) Only one base changes normally third base 3)Explain what a mutagen is a) Radiation or chemicals that increase mutation rates in DNA b) Chemical mutagens includei) Nucleotide base analogs resemble DNA bases, increase the rate of substitutionsii) DNA modifying chemicals cause alkylations, nitrosations, hydroxylationsiii) Intercalating agents bind between DNA bases causing insertions and deletionsiv) Radiations: UV light, ionizing radiations. Mutations repair may lead to erroror deletion4)Compare and contrast same-site and second-site reversions a) Same site reversioni) The mutated base pair mutates back to original genotypeb) Second site mutationi) Mutation at a different site restores the original phenotype(1) Example: A second frame shift mutation restores the correct reading frame5) Explain why deletions and insertions can have dramatic effects on protein sequencesa) Frame shifts cause changes in all amino acid codons downstream of the mutation siteb) The reading frame is changed and often have severe effect on protein function6) Explain how RecA and LexA regulate each other in the SOS repair pathway a) RecA- activated by DNA damageb) LexA- Repressor of many SOS genesc) Activated RecA degrades LexA DNA repair genes are no longer repressed7) Explain what the Ames test is used for a) Measures the mutagenicity of potentially hazardous chemicals8) Explain how the Ames test is performed a) The Ames test measures the rate of reversion mutationsi) The bacterial strain used contains a point mutation in a gene involved in histidine synthesisii) Bacteria plated on agar growth medium containing no histidine(1) Only revertants will growiii) Disk infused with the tested chemical in the centeriv) Increased number of colonies around the disk indicates a positive Ames test result (chemical is a possible mutagen) 9) Explain how to interpret its results a) If there are an increased number of colonies around the disk then the Ames test is positive and that chemical is a possible mutagen10) Explain why the Ames test measures the rate of reversion mutations, not first-hand mutations a) Testing if chemical is a mutagen. If chemical is a mutagen revertants will grow because have mutated back to original genotype or phenotype so have functional protein. b) Cant use to test first hand mutations because if it is a mutagen many proteins will become nonfunctional and bacteria won’t grow11) List and order the steps in the homologous recombination pathway a) Definition- DNA segments have nearly the same sequence b) Steps:i) 1. Endonuclease nicks donor NDA (cut only one of the 2 strands)ii) 2. Nicked strand detaches from other strand and binds single stranded binding proteinsiii) 3. Strand invasion: nicked strand displaces homologous region of recipient DNA (withassist from RecA protein)iv) 4.Crossed strand exchange: formation of crossed strand structure (“crossover” or “Holiday junction)v) 5. Resolution: Cutting of DNA strands yields new DNA with heteroduplex regions- segments originating from different DNA molecules12) Mention important proteins involved in recombinationa) SSB proteinb) RecA protein 13) Explain what heteroduplex DNA is a) Segments originating from different DNA molecules14) Compare and contrast transformation and transduction a) Transformation i) A horizontal genetic transfer process where external free DNA is taken into a recipient cellb) Transduction i) Horizontal gene transfer process during which a phage transfers DNA from a donor cell to recipient cell15) Compare and contrast generalized and specialized transduction a) Generalized i) A random piece of chromosomal DNA accidentally is packaged into a phage and canbe transferred to a recipient cellii) Transducing particles are not infectiousiii) Any gene in the genome can be transferred, but at very low frequencyb) Specializedi) Only genome DNA adjacent to the prophage can be transferred to a recipient cellii) Only happens with lysogenic phagesiii) More frequent than generalized transduction iv) After incorrect excision of the prophage every phage has a small piece of chromosome (a small piece of phage DNA may be left behind) This DNA can be injected into a recipient cellv) The transferred donor DNA can then be recombined in the genome of the recipient cell16) Explain what type of phages cause specialized transduction a) Lysogeny- Phage DNA intergrated into the chromosomeb) Only lysogenic phage17) Explain what natural competence is a) Certain bacteria have specific proteins to take up external DNA18) Explain how bacteria that are not naturally competent can be transformed a) The must be able to take up external DNAb) Transformation involves uptake of external DNA and integration of the DNA by homologous recombination19) Compare and contrast vertical and horizontal gene transfers a) Vertical i) Refers to the inherited genes passed to new cells as a result of cell divisionb) Horizontali) The lateral, non-inherited transfer of genes from one cell to another(1) Several mechanisms: transformation, transduction, and conjugation(2) Detected by careful analysis and sequence comparisons of
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