MMG 301, Lec. 25Mutations and BacteriophageQuestions for today:1. What are mutations and how do they form?2. How are mutant bacteria used in research?3. What are the general properties of bacteriophage (viruses of bacteria)?4. How do phage grow? 5. How do we assay for phage?Overview of mutationsDefinitions:Mutation: an inheritable change in the base sequence of a genomeMutant: organism carrying a mutationGenotype:genetic makeup of an organismPhenotype: observable characteristics of an organism (pigments, overall appearance such as flagella or capsule, nutritional growth requirements, etc.)Types of Mutations:Base pair changes and insertions/deletionsPossible base pair changes to a codon for TyrosineThis figure shows the insertion or deletion of a single base, but larger segments can be inserted or deletedShifted reading frames are likely to result in the formation of stop codonsEffects on the cell (phenotype):No effect: this is always the case for silent mutations, often seen with missense mutations, and may occur with insertions/deletions that retain the same reading frame.Death:this can be hard to study! One approach to examine such mutants is to include 2 copies of the gene, where only one is mutated.Conditional lethality: This is the best phenotype for genetics studies. Cells survive only under a subset of conditions- New growth requirement: e.g., mutations in a trp gene will prevent cell growth unless tryptophan (Trp) is provided (the mutant is an auxotrophwhereas the wild-type cell is referred to as a prototroph).- Unique temperature dependence: e.g., the mutation alters an enzyme needed for bacterial survival. The variant enzyme works in cells grown at 30ºC, but is inactivated in cells grown at 37ºC.Causes of mutations:Errors in replication: Spontaneous. Occurs at a rate of 10-7to 10-11per base pair. For a 1000-bp gene, this means 10-4to 10-7chance of a mutation per generation. If 108cells/ml will likely find mutant!Chemical mutagens:- Chemicals that modify DNA. Nitrous oxide deaminates A and CHydroxylamine reacts with CEthylmethane sulfonate adds CH3group(repair enzymes exist!)Nitrosoguanidine crosslinks 2 DNA strands- Intercalators insert into stacked bases to resemble an extra base. Example = ethidium bromide. This hazardous chemical is frequently used in labs to visualize DNA in gels due to UV fluorescence.- Base analogs resemble true bases, are incorporated into DNA, but lead to incorrect base pairing.Radiation- UV light causes thymine dimerizationrepair enzymes exist- X-rays/cosmic rays/gamma rays lead to production of hydroxyl radicals (OH•) that can damage DNA in several ways including strand breaks. (Repair enzymes exist. Deinococcus radiodurans has special proteins that hold DNA together to allow repair)Biological agents- transposons are DNA fragments that “jump” from one position to another using inverted repeats (IR). They will cause a mutation if they land inside and disrupt a gene. They are likely derived from viruses.IR- viruses can also disrupt genes if they become inserted into a genome (see prophage below)HNNNNHOOCH3CH3OORRWorking with mutant bacteriaHow do we isolate a desired mutant? Example = Trp auxotrophSpread a sample of mutated culture on complete medium (A, containing Trp) at a dilution to give individual colonies.“Replicate plate” the colonies onto (A) and (B) containing medium lacking Trp.Colonies growing on A, but not B, are likely Trp auxotrophs defective in a trp gene.Penicillin selectionThe above method works great if you have lot’s of mutants, but this is usually not the case. A useful method to “enrich” for mutants is by penicillin selection.A culture is inoculated into liquid medium lacking the growth factor of interest (e.g., Trp) and containing penicillin. Cells able to grow without Trp are killed by penicillin, whereas the auxotrophs cannot grow so are not affected by the antibiotic. After transfer to a medium without penicillin, but with Trp, the mutants grow.CellsMedium A-Trp+ penicillinMedium B+Trp- penicillinEnriched for TrpauxotrophsUsing mutants to test for mutagens:Ames test: his auxotroph requires His for growth. The mutation spontaneously “reverts” so that ~10 of 108cells can grow without His. Mutagens (added to a paper disk) increase the reversion frequency.[Next lecture: using mutants to study gene transfer]General Properties of BacteriophageDefinitionsBacteriophage: A virus or genetic element containing DNA or RNA that requires a host bacterial cell for replication, but has an extracellular state.Virion: virus particle.StructuresToo small to see by light microscopy, can only visualize by electron microscopy.Naked phage (more common) contain the genetic material surrounded by a protein shell (capsid) composed of capsomer proteins. The capsid plus genetic material is the nucleocapsidEnveloped phage additionally possess an envelope derived from the host.Types of nucleic acid depend on the phage, with dsDNA (48,514-bp Lambda or 168,903-bp T4), ssDNA (ΦX174 of only 5,386-bp), dsRNA (φ6), orssRNA (MS2) known.In the case of ssRNA, the strand may be the coding (+) sense (i.e., mRNA) or the opposite (-) sense. The method of replication/transcription will depend on the type of nucleic acid:dsDNAssDNAssRNA(+) ssRNA(-) dsRNAEukaryote viruses(AIDS)Properties of the capsid: Made of one or a few types of capsomer proteins that spontaneously assemble into various defined shapes.- helical- icosahedral- complexAll the same protein, or distinct proteins in different parts of the structure (proteins A vs. B)The capsomer also often carries phage-encoded enzymes made by the host cell. These enzymes may include phage-specificpolymerases and nucleases to degrade the host nucleic acidsGrowth cycle of phageAttachment: highly specific due to phage proteins binding to a host cell receptor (protein, carbohydrate, lipid, etc.). The receptor is not designed to bind phage, rather it has a normal cellular role (e.g., Fe transport).Penetration: Nucleic acid (and some proteins) enter cell, whereas capsomer often remains external. In the case of some phage (e.g., T4) nucleic acid is injected into the cell.OM/LPSpeptidoglycanIMEarly infection: early mRNA transcribed, early proteins made to begin replication process.Replication: Phage nucleic acid is synthesized.Viral protein synthesis: capsomer components formed.Assembly: spontaneous formation of new phage.Release:- in some cases, new phage “leak” out without killing the host
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