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BIOL 140 Module 6 Notes.docx

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BIOL 240
Trevor Charles

MODULE 6 Part A: An Introduction to the Terminology of Molecular Biology History - E. coli and Salmonella  developed in first genetic system Important Terms - Mutant: strain carrying mutation - Mutation: change that occurs within gene to make another allele of gene or disrupt function of gene - Mutation – gain/loss of function, change of function at DNA level - Genotype: description of all alleles within organism - Phenotype: observable properties of strain containing mutation - Wild-type: strain found in nature  original isolate of strain that all mutants are derived from Part B: The Phenotypes of Mutations – Selection vs. Screening Selectable Phenotype - Bacteria plated out on growth medium under certain conditions that will allow phenotype of interest to grow - Power of genetics  ability to use selection vs. screening - Screenable phenotype – cannot be selected but can be differentiated Isolation of Mutants - Error occurring during replication  leads to evolutionary change - Mutations arise spontaneously Replica Plating - Culture of wild-bacteria subjected to mutagenic conditions to increase mutation rate - Culture diluted and placed on complex medium tor single colonies on master plate - Auxotroph: organism that developed nutritional requirement through mutation - Potential auxotrophs do not grow on minimal medium Part C: Types of Mutations Examples Auxotrophs - loss of enzyme in biosynthetic pathway - inability to grow on medium lacking nutrient Cold-Sensitive - inactivated protein at low temperature - inability to grow at temperature normally supporting growth Drug-Resistant - Permeable to drug - Growth on medium containing growth-inhibitory concentration of drug Noncapsulated - Loss or modification of surface capsule - Small rough colonies and not large smooth ones Nonmotile - Loss of/non-functional flagella - Compact colonies instead of flat, spreading ones Pigmentless - Loss of enzme in biosynthetic pathway leading to loss of pigment(s) - Presence of different/lack of colour Rough Colony - loss or change in lippolysaccharide outer layer - Granular/irregular colonies Sugar Fermentation - Loss of enzyme in degradative pathway - Lack of colour changes on agar with sugar and pH indicator Temperature Sensitive - alteration of essential protein so becomes more heat-sensitive - inability to grow temperature normally supporting growth Virus Resistant - loss of virus detector - growth in presence of large amounts of virus Shifts in Reading Frame - deletion or insertion mutation - To avoid shifs in reading frames  add or delete 3 amino acids Part D: Mutagens - Mutagens can increase frequency of mutations The SOS Response - several genes with expression induced to respond to DNA damage - Some genes can repair damage - sometimes DNA damage is so severe cell dies if not repaired - UvrA protein  error-gree repair of DNA damage - UmuD protein  error-prone repair of severe chromosomal damage - SOS response – last resort of the cell - RecA detects DNA damage  lexA (binds operator in front of gene) cleaved - Single stranded DNA = signal to RecA that DNA damage has occurred Part E: The Ames Test Test for Mutagenicity - determines whether product or chemical is mutagenic - if mutagenic, possibly carcinogenic - Salmonella strain containing point mutation in histidine biosynthetic pathway is used - Salmonella strain = histidine auxotroph - measures rate of mutation Part F: Genetic Recombination Introduction - DNA transferred from one bacterial strain to another - Transformation: transfer of DNA between bacterial strains - Transduction: involves use of bacterial phage to transfer segments of chromosome - Plasmids also involved in transfer of genetic material The Mechanism of Homologous Recombination - Recombinatoin – event allowing DNA molecules to be joined - Occurs in multicellular eikaryotic organisms - High level of recombination during meiosis - Can also occur in bacteria when segments of DNA inserted from one strain to another - Segments have different alleles  exchange of alleles between organisms - Single stranded donor molecule base pairs with recipient molecule (RecA) - strand bound to recipient displaced by donor molecule - Cross-strand exchange develops with formation of Holliday junction - Splicing of donor and recipient  pathces or splices recombination DNA Transfer - Donor  free DNA liberated out of cell - Taken up by Recipient cell - Transformation: transfer of DNA - Transduction: transfer of host genes from one cell to another by virus - Conjugation: transfer of genes from one prokaryotic cell to another by cell-to-cell contact - Plasmid: extrachromosomal genetic element that is not essential for growth and has no extracellular form - Plasmid transferred through conjugation Detection of Recombination - selectable markers or phenotypes  determine whether recombination has occurred - ability to select cells out of background – selectable phenotype Transformation and Transduction Transformation - active process involving number of genes which encode proteins that mediate transformation event - Nuclease  single stranded DNA  attracts RecA therefore very recombinogenic Generalized Transduction - virus may accidentally package host DNA into bacteriophage - Phage particles and tranducing particles are identical in composition  different DNA - cannot function as phage when infection new cell  different DNA - Alleles in new DNA differs from exisiting DNA – genetic change - Recombination required Specialized Transduction - occurs in bacterial viruses where part of viral life cycle includes integration of viral DNA with bacterial chromosome - Phage lambda – phage that integrates with E. coli DNA at site of galactose genes - Recombination at wrong place  phage DNA remains behind and some bacterial DNA is incorporated into phage (excised DNA) Transducing Particles in Generalized Transduction and Specialized Transduction - Specialized transduction  every particle = transducing particle - Each specialized transducing particle  contains phage DNA - Generalized transducing particles never contain phage DNA  only host DNA - Specialied transducing particles originate after aberrant recombination event - Generalized transducing particles originate after mistake in packaging where host chromosomal DNA and phage DNA never connected Plasmids The F Plasmid - Conjugation carred out by plasmids in bacteria - “F”  fertility - Allows conjugation and gene transfer to occur between donor and recipient - Genes expressed in tra region to generate pilus, make enzymes, etc - DNA polymerase initiates replication of plasmids to maintain certain number of copies within bacterial cell - regulation of replication ensures correct number of copies - Insertion sequences and transposons  related genetic elements - jump
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