3/4-3/7 Notes: Mutations
3/4-3/7 Notes: Mutations Bios 312
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This 3 page Class Notes was uploaded by Cara Cahalan on Wednesday January 13, 2016. The Class Notes belongs to Bios 312 at University of Nebraska Lincoln taught by Karrie Weber in Spring 2016. Since its upload, it has received 22 views. For similar materials see Microbiology in Biology at University of Nebraska Lincoln.
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Date Created: 01/13/16
3/4 3/7: Microbial Genomics: Mutations 10.110.4 Readings: 6.16.4, 6.106.13 I: Investigating Genomics 6.1 Introduction to Genomics Genomics mapping, sequencing, analyzing, and comparing genomes Bacterial genome range: 0.513 Mbp. Human genome 3 million base pairs 6.2 Sequencing Genomes Sequencing precise order of subunits in a macromolecule, order in which the nucleotides are aligned Generations of DNA sequencing: o First gen dideoxy method, Fred Sanger Dideoxynucleotides to block chain extension, and using labeled precursors for detection DNA primers used (more stable than RNA primers) Copied ssDNA and dideoxynucleotides are added for each base. Analog is a chain termination reagent (lacks 3’ –OH), inserted randomly Shotgun sequencing preparation of DNA for sequencing, genome cleaved and cloned. Detect overlapping sequences and assembled into correct order o Second gen massively parallel methods Samples are sequence side by side, requires increased computing power (100x faster) Each time dNTPs energy released for lucrifase (lightemitting enzyme) o Third gen sequences single DNA molecules, needs two model features: Allows detection of single flashes of light for single nucleotides Fluorescent tags attached to the pyrophosphate is discarded burst of color o Fourth gen post light sequencing measures release of protons when a new dNTP is added Pore detector DNA moves through and electrical changes indicate the type of nucleotide Genome assemblyputting fragments in correct order and eliminating overlaps Annotation identifying genes and other functional regions in the genome Closed genome entire genome sequence is determined, more expensive and require more human input 6.3 Bioinformatics and Annotating Genomes Bioinformatics storage and analysis of structures of nucleic acid and proteins in the genome Microbial genomes consist of ORFs separated by short regulatory regions and transcriptional terminators Functional ORF encode a protein To find an ORF, need to identify the start and stop codons in each sequence II: Microbial Genomes 6.4 Genome Size and Content Comparative genomics compares genomes based on size, organization, and gene content Each Mbp of DNA in prokaryotes encodes about 1000 ORFs o As prokaryotic genomes increase, gene number increase proportionally (not true of Eukaryotes introns) Autotrophs need fewer genes than heterotrophs Eukaryotes have large introns, prokaryotes do not, more genes than eukaryotic genomes despite having less DNA Genomes are modeled by adaptation to an organisms life Comparative analysis help identify genes that code for enzymes Genes of protein synthesis overtake metabolic genes on a percentage basis as genome size decrease Proteins involved in: o Core cellular processes minor variations in gene number and genome size o More specialized more variation Larger genomes are able to have more specialized metabolic genes due to increased competition in environment 6.10 Metagenomics Metagenomics analyzes pools of RNA or DNA from environmental sample o Metagenome gene content of the organisms inhabiting a certain environment (most are viruses) Microbiome collection of prokaryotic cells in humans IV: Evolution of Genomes 6.11 Gene Families, Duplications, and Deletions Homologs genes that are related due to shared evolutionary ancestry Paralogs related genes due to gene duplication in evolution of organism Orthologs genes found in one organism that are similar to another because of a common ancestor After duplication one duplicate can evolve and other supplies cell with original function microbial evolution 6.12 Horizontal Gene Transfer and Genome Stability Horizontal gene transfer transfer of genetic material from one cell to another o 3 mechanisms: transformation, transduction, and conjugation o Horizontally transferred gens encode metabolic functions and virulence genes 6.13 Core Genome versus Pan Genome Core shared by all strains of a given species, typical of species as a whole Pan core plus optional extras present in one or more strains, but not all, include mobile elements of some strains Chromosomal islands extra blocks of genetic material in chromosome, contain noncrucial specialized functions o Presumed to have foreign origin because: Flanked by inverted repeats whole region was inserted into a chromosome Base composition/codon bias are different than the genome proper Found in some strains of a species and not others o Encode virulence factors (special proteins that help initiate disease) on pathogenicity islands Lecture: Mutations: o Induced those made deliberately o Spontaneous occur without human intervention, exposure to natural radiation or oxygen radicals Base pair substitution: o Point mutation change only one base pair, can lead to single amino acid change in protein or no change Normal protein wild type Silent does not affect amino acid sequence Nonsense codon becomes stop codon, polypeptide and protein is incomplete Missense amino acid changed, polypeptide altered, faulty protein formed o Reversible through a reversion alteration in DNA that reverses effects of prior mutation Revertant strain in which original phenotype that was changed in the mutant is restored Samesite restored at same site as original mutation Secondsite mutate different site, compensating for the effect of the original mutation o Frame shift: deletion/insertion that result in shift in reading frame, result in complete loss of gene function Insertion +1, deletion 1 Deletions do not revert, insertions can revert if a subsequent deletion alters reading frame Transitions one purine base (A/G) is substituted for another purine; or one pyrimidine base (C/T) for another o AG GA or CT TC Transversions point mutation where purine base is substituted for a pyrimidine base, vice versa Mutagenesis: spontaneous mutation frequencies 6 7 o 10 to 10 per kilobase pair during a single round of replication o Mutation rates in an RNA genome are higher because comparable RNA repair mechanisms don’t exist o Perfect fidelity in organisms is counterproductive prevents evolution Mutagens: chemical, physical, or biological agents that increase mutation rates o Chemical: Nucleotide base analogs: resemble nucleotides Chemical mutagens that induce chemical modifications or cause frameshift mutations o Physical: (electromagnetic radiation) Nonionizing (UV radiation) purines and pyrimidines strongly absorb UV Ionizing (i.e., Xrays, cosmic rays, and gamma rays) Produce free radical that damage macromolecules in the cell Ames Test: bacterial mutations detect for potentially hazardous chemicals o Looks for an increase in mutation of bacteria in the presence of suspected mutagen DNA repair systems o Direct reversal: mutated base is still recognizable and can be repaired without referring to other strand o Repair of single strand damage: damaged DNA removed and repaired using opposite strand as template o Repair of double strand damage: a break in the DNA, requires more errorprone repair mechanisms o SOS system regulon, repairing dsDNA proceed without template, more error prone. When DNA damage is large scale, the cell may use a different type of repair system Allows replication to proceed and cell to replicate Translesion synthesis allows DNA to be synthesized with no template Two proteins regulate: LexA (repressor) RecA (activated in presence of DNA damage) Selectable and nonselectable mutations: o Selectable mutant has growth advantage under certain environmental conditions Useful in genetic research, mutant capable of growing on culture containing previous mutagen o Nonselectable mutationshave neither an advantage nor a disadvantage over the parent Screen is more tedious than selection, methods that facilitate screening o Replica plating identifies cells with growth requirement Master plate with growth medium pressed onto velveteen which is imprinted of all colonies Complete and minimal mediums, transfer imprint of colonies to fresh media All colonies grow, mutants do not grow Key Concepts: Mutations can arise spontaneously or as a result from exposure to a mutagen These mutations can have no effect, positive effect, or negative (cell death) Perfect fidelity in organisms is counterproductive because it prevents evolution
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