Microbiology Week 6 Lecture Notes
Microbiology Week 6 Lecture Notes 101.0
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This 6 page Class Notes was uploaded by Isabel Markowski on Friday October 16, 2015. The Class Notes belongs to 101.0 at University of Wisconsin - Madison taught by a professor in Fall 2015. Since its upload, it has received 46 views. For similar materials see General Microbiology in Microbiology at University of Wisconsin - Madison.
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Date Created: 10/16/15
MICROBIAL GENETICS PART 2 Basics Genome Structure 0 Genome All genetic material chromosomes and accessory DNA pieces 0 Typically have one chromosome and one plasmid o Chromosome 100012000 kb carry essentiaI genes Kb kilobase 1000 base pairs 0 Plasmid 3500 kb I Completely different DNA than chromosomes I Much smaller I Nonessential genes but beneficial ex antibiotic resistance nutrient degradation virulence functions I DIPSENSABLE I 1000 s different plasmids I Have ori origin for replication DNA Replication o Held together by Hbonds 0 Separation of DNA strands to daughter DNA molecules each one one parental strand and one new 0 Sequence determined by base complementarity Bacteria Pmtems o DnaA binds to oriC and recruits other proteins 1st step in replication 0 Circular chromosomes 0 Replication bidirectional 1 DNA opened unwound and primed help polymerase in step 2 o Helicase unwindsopens strands o SSBP SingleStrand DNAbinding proteins stabilize single strands o Primase makes RNA primer 0 Topoisomerase relieves twisting forces unwinds tension 2 Synthesis of leading strand begins 0 DNA Polymerase makes new DNA I Needs primer to start and adds new nucleotide to 3 end only 5 to 3 direction 0 Both strands being worked on at same timerate 0 Leading strand and lagging strand 0 Replisome set of proteins 0 Both RNA transcription and DNA replication polymerases synthesize nucleic acid in 5 to 3 direction 0 But DNA polymerase requires primer to begin Errors 0 DNA Replication is NOT perfect 0 Error Rate characteristic for organism o Determined by I DNA Polymerase accuracy during synthesis I Proofreading many DNA Polymerases correct their mistakes during replication DNA polymerase III noticescorrects if adds mismatched base pair 0 Errors gt Mutations o Heritable change in nucleotide sequence of genome 0 Errors during DNA Replication about 1 nucleotide in 108 incorrect o Caused by I Spontaneous chemical changes in bases I Mutagens agents that damage DNA chemicals UV radiation etc o Impacts I Change in genotype nucleotide sequence of genome I Does it change phenotype observable properties of organismphysical I Impact can be neutral detrimental or beneficial depends on what s encoded 0 Loss of function quotknockoutquot 0 Altered function 0 Gain of function ex antibiotics Silent Mutation point Mutation Changing Single peint in sequence Missense Point Mutation 0 Nucleotide pair substitution silent 0 Change amino acid sequence 39 WI only affect genotype Nonsense Point Mutation Puts in stop codon Insertionsdeletions 0 Big impact translation stops Insertion frameshift Knockout effect 0 Insert extra 0 Scrambles message 0 Knockout effect Deletion frameshift o Deletes 0 Changes reading frame 0 Knockout effect o Uncorrected errors genetic change in daughter cells quotWild Type mother cell base pair mismatch 0 DNA Repair systems Daughter Cell like original DNA Mutant Daughter Cell changed base pair 0 Set of enzymesremove errors and insert correct sequence 0 Ex Nucleotide Excision Repair scan for problems cut nucleotide replace it repair linkage bonds 0 Horizontal Gene Transfer 0 Three types Transformation Uptake of free DNA and incorporates into own Natural Transformation some microbes naturally able to take up foreign DNA streptococcus heisseria spp bacillus subtilis Artificial Transformation lab can induce many species 0 Called quotcompetencequot 0 Historical importance streptococcus pneumoniae and the transforming principle Lead to evidence that genes made of DNA 0 S smooth produces capsule virulent disease causing o R rough no capsule nonvirulentdoesn t cause disease 0 Heatkilled dead S cells can still kill animals in combination with R cells because of transformation when S cells killed DNA liberated by heat and can I Capsules important 0 Involves celltocell contact Transduction Bacterial virus moves DNA cell to cell Generalized transduction random packaging of host chromosomal fragments Viral DNA injected and genes copied when replication lytic cycle Transduction transducing particle contains donor cell DNA 0 Homologous recombination 0 Chromosomal not viral DNA injected no virusescell death Conjugation 0 Some plasmids encode transfer machinery for their transfer to another cell 0 Requires DNA export system and pili attaches to other cell and reels in o Requires two live cells 0 Can transfer DNA bn widely different species less specific 0 Microbial genomes can change by mobile DNA dynamic I All species have core genome some have pan genome variable even within species I 2 strands same species can vary greatly 0 Very commonBacteriaArchaea less in Eukaryotes 0 Can transfer across laterally to other living cells vs down through reproduction Innate Immunity RestrictionModification Systems 0 Quality check 0 REase restriction endonuclease degrades o MTase DFNA methyltransferase marks cell self 0 Occurs during all three types of gene transfer 0 Cell produces enzymes restriction modification complex chemically modify DNA Recognition Site foreign nonself Methylated recognItIon site marked as self REase MTase 0 Hundreds of restriction enzymes 0 Viral response types to restrictionmodification system how to overcomedefeat systems to infectsurvive 0 Use of unusual nucleotides modify geneshide 0 Use of DNA binding proteins protectionshield o Stimulation of methylation try to beat out speed of restriction enzymes 0 Degradation of restriction enzymes Microbial Genomics 0 Look at all of genes together and interpret 0 Use computer programs 0 Find coding sequences ORF s open reading frame 0 ORF s potential protein to be made 0 Computers look for ORF s for potential to make protein on both DNA strands Find similar protein sequenceshelp suggest function Bacterial and archaeal genomes efficient infodense 0 Almost all of DNA codes for proteins 0 Vs eukaryotes full of noncoding DNA Microbial genomes vary even win species diversity APPLED GENETCS PCR Amplification of DNA 0 PCR polymerase chain reaction procedure to selectively amplify piece of DNA 0 Involves quotin vitro DNA synthesis make copies of specific DNA section 0 Can amplify from tiny amounts of template DNA very sensitive 0 Components DNA template strand primers DNA polymerase dNTP s deoxynucleotide triphosphates Process 0 PCR mix in a PCR machine thermocycler o Denaturation 0 Heat 9495 C for 30 seconds 0 Strands separate helicase unwinds DNA 0 PCR primers must bind to sequences on either side of target sequence on opposite strands Primer 5 3 339 Y 5 Primer o Annealing 0 Cool 55 C for 30 seconds 0 When target DNA is singlestranded primers bind anneal and allow DNA polymerase to work S39 y Prlmer 3 5 Primer p 539 339 5 3i 0 Extension 0 72 C for 60 seconds per kb of target 0 Repeat 0 Usually 30 cyclesto get many copies 0 Because polymerases would be destroyed by heat in denaturation step and you d have to keep putting polymerases back in after each cycle found different thermostable polymerases from microbes o Taq polymerase I Thermos aquaticus thermophilic bacterium from hot springyellowstone I Grows at temperatures of up to 90 C I Can use in PCR process I Lacks proofreading ability o Vent polymerases I Thermococcus Iitoralis hyperthermophilic archaeon isolated from submarine thermal vent I Proofreading ability higher fidelity and stability 0 Deep vent polymerase I Pyrococcus sp Strain GBD hyperthermophilic archaeon isolated from submarine thermal vent I Able to grow at temperatures 104 C I Proofreading higher fidelity and stability
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