Exam 2 Study Guide
Exam 2 Study Guide 4000
Popular in Basic and Practical Microbiology
Popular in Microbiology
This 60 page Study Guide was uploaded by Victoria Gonzalez on Monday October 26, 2015. The Study Guide belongs to 4000 at Ohio State University taught by Madhura Pradhan,Tammy Bullwinkle in Summer 2015. Since its upload, it has received 147 views. For similar materials see Basic and Practical Microbiology in Microbiology at Ohio State University.
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Date Created: 10/26/15
Chapter 5 Control of Microbial Growth Victoria Gonzalez Terms Sterile completely free of all viable microbes an absolute term Sterilization destruction or removal of all microbes through physical or chemical means Sterilant a chemical that destroys all microbes Disinfectant a chemical that destroys many microbes Decontamination treatment used to reduce the number of pathogens to a level considered safe Sanitation the process that substantially reduces the microbial population to meet accepted health standards Antiseptic disinfectant nontoxic enough to be used on skin Aseptic technique procedures that minimize the chance of unwanted microbes being accidentally introduced Pasteurization brief heat treatment that reduces the number of spoilage organisms and destroys diseasecausing microbes Germicide kills microbes Preservation process of inhibiting microbial growth to delay spoHage Bactericidal kills bacteria Bacteriostatic prevents the growth of but does not kill bacteria Decimal reduction time Dvalue time required for 90 of the organisms to be killed under speci c conditions Things to know Basic principles and terms associated with microbial growth control What to consider when choosing a method and why The different physical methods for killing microbes and basics of how each work types of heat pressure ltration radiation 4 levels of Chemicagermicide potency What to consider when choosing a chemical method of microbial control 10 classes of germicidal chemicals examples pros cons and uses Rising concerns about germicide resistance and toxicity Different methods of preservation 1 History a Joseph Lister i Inspired by Pasteur s work wondered if minute organismsquot might be responsible for causing infection ii Provided additional support for germ theory iii Began sterilizing instruments and maintaining a clean operating environment b Prior to the late 19th century minor surgeries were risky for the patient to develop fatal infections i Proper hand washing and gloves were not standard ii Did not understand that airborne microbes could infect open wounds c Modern hospitals i Use strict procedures to avoid microbial contamination ii Most surgeries can be performed with relative safety d Control of microbial growth is important in i Preserving food and other goods ii Control practices in research labs iii Public spaces and utilities need to ensure safe levels of microbes drinking water waste water air ltration 2 51 Approaches to Control a Basic principles i Sterilization removal of almicroorganisms 1 Includes endospores and viruses not prions ii Disinfection elimination of most or all pathogens 1 Some microbes may remain iii Disinfectants used on inanimate objects iv Antiseptics used on living tissues v Pasteurization brief heating to reduce the number of spoilage organisms by destroying pathogens 1 For foods and inanimate objects 2 Pasteur found that brief heating beer and wine prevented the growth of most microorganisms that cause food spoilage 3 Different levels of pasteurization 72138 C vi Decontamination reduces pathogens to levels that are considered safe to handle vii Sanitation process that substantially reduces microbial population of meet accepted health standards 1 Not a speci c level of control vague term viii Preservation delay spoilage of foods and other perishable products by adjusting conditions or adding bacteriostatic growthinhibiting preservatives b Situational considerations different situations need different levels of microbial growth control i Daily 1 2 ii 1 2 3 4 5 iii Micro 1 2 3 iv Food 1 life Washing and scrubbing with soaps and detergents a Mechanicacleaning Refrigeration preservation Hospitals and healthcare facilities Important due to healthcare associated infections Patients are susceptible to infection Pathogens are everywhere Instruments must be sterilized to avoid infection Prions are a new concern dif cult to destroy biology laboratory Rigorous methods of control sterilization and disinfection Must eliminate microbial contamination to experimental samples and the environment Careful treatment before sterile media and after sterilize cultures waste Aseptic techniques are used to prevent contamination of samples self and laboratory CDC guidelines for labs working with microbes a BSL l microbes not known to cause disease b BSL 4 microbes for which no vaccinetreatment exists i BSL biosafety level production Perishables retain quality longer when contaminating microbes are destroyed removed or inhibited Heat treatment is common and reliable but can alter avour and appearance of products Irradiation approved to treat certain foods Chemical additives prevent spoilage FDA regulates them because of toxicity risk Facilities must keep surfaces clean and free of microbes v Water treatment 4 5 Goal ensure that drinking water is free of pathogens Chlorine was traditionally used to disinfect water a It forms disinfection byproducts DBPs when reacting with naturally occurring chemicals b DBPs are linked with longterm health risks Some organisms are resistant to chemical disinfectants a C parvum which causes diarrhea Regulations require facilities to minimize DBPs and C parvum in treated water Filtration UV or ozone is often used c The selection of effective procedures can be complicated i The ideal method does not exist ii Each method has drawbacks and procedural parameters iii Must weigh risks and pick the best procedure for the job 3 52 Selection of an Antimicrobial Procedure a To pick a procedure for microbial control you must consider i The type and number of microbes present ii Environmental conditions Risk of infection iv The composition of the item b Types of microbes i Highly resistant microbes 1 2 Bacterial endospores only extreme heat or chemicals completely destroys them Protozoan cysts and oocysts resistant to disinfectants excreted in feces causes diarrheal disease if ingested Mycobacterium species waxy cell walls make the bacteria resistant to many chemical treatments Pseudomonas species resistant to and can grow in some disinfectants form bio lms hard membrane Naked virus lack lipid envelope more resistant to disinfectants ii Intrinsic resistance resistance of an organism to an antimicrobial medication due to the inherent characteristics of that organism c Number of microorganisms takes time for heat and chemicals to kill time is affected by the population size i A fraction of microorganisms dies after a given time 1 Larger populations require more time 2 Removing organisms by washing reduces time 3 Decimal reduction time D value time required to kill 90 of the population under speci c conditions a Gauges commercial effectiveness b Subscript is used to indicate temperature D120 d Environmental conditions i Dirt grease and body uids can interfere with heat penetration and the action of chemicals ii It is important to Clean thoroughymechanical efforts iii pH and temperature can in uence effectiveness of chemicals 1 Bleach kills faster at 55 C 2 Bleach is more effective at a low pH e Risk of infection i Medical instruments are categorized according to risk for transmitting infectious agents 1 Critical items come in contact with body Ussues a Must be sterile b Needles scalpels forceps 2 Semicritical instruments contact mucous membranes but do not penetrate tissues a Must be free of virus and vegetative bacteria b Few endospores left are blocked by mucous membranes c Endoscopes and endotracheal tubes 3 Noncritical instruments contact unbroken skin only a Low risk of transmission b Countertops stethoscopes blood pressure cuffs f Composition of item i Some sterilization and disinfection methods are inappropriate for certain items 1 Heat is inappropriate for plastics 2 Irradiation provides an alternative but damages some types of plastic 3 Moist heat liquid chemical disinfectants etc cannot be used to teat moisturesensitive materials 4 53 Using Heat to Destroy Microorganisms and Viruses a BSDOFT Heat is very useful safe no toxins reliable fast and inexpengve Heat can be used to sterilize or disinfect Moist heat irreversibly denatures proteins Dry heat less effective Boiling water at 100C i Boiling for 5 min destroys most microorganisms and viruses ii Useful for treating drinking water iii Does NOT sterilize endospores can survive Pasteurization i Signi cantly reduces the numbers of heat sensitive microorganisms ii Destroys pathogens and spoilage organisms iii Hightemperature shorttime HTST most products 1 Milk 72C for 15 seconds 2 Ice cream 82C for 20 seconds iv Ultrahightemperature UHT shelfstable boxed juice and milk 1 Called ultrapasteurization 2 Milk at 140C for a few seconds then quickly cooled g Pressurized steam i Autoclave used to sterilize using pressurized steam 1 2 3 4 5 Increased pressure raises temperature kills endospores Sterilization is typically at 121C for 15 minutes a Longer for larger volumes Flash sterilization at higher temperature can be used Prions are thought to be destroyed at 132C for 1 hour Routinely used for laboratory medias glassware medical equipment surgical instruments commercial canning h Commercial canning uses an industrial sized autoclave called retort i Designed to destroy C botuinum endospores ii Otherwise endospores can germinate in canned foods because cells grow in lowacid anaerobic conditions and produce a toxin iii Canned food is considered commercialy sterile 1 Endospores of some thermophiles may survive not a concern because they only grow at temperatures well above normal storage temperatures i Hot air ovens oxidize cell components denature proteins i Useful for powders oils and other dry materials j Incineration is a method of dry heat sterilization i Oxidizes cell to ashes ii Used to destroy medical waste and animal carcasses iii Laboratory inoculation loop is sterilized by aming 5 54 Using Other Physical Methods to Remove or Destroy Microbes a Filtration useful for removing microbes from large volumes of liquid or air also some uids cannot withstand heat treatment i Filtration retains bacteria ii Filtration of uids is used extensively 1 Membrane lters a Small pore size thin 2 Depth lters a Thick porous ltration material cellulose b Larger pores c Electrical charges help trap cells iii Filtration of air 1 High ef ciency particulate air HEPA lters remove nearly all microbes from the air 2 Useful for specialized hospital rooms and laboratory hoods 10 b Radiation i The type of radiation will determine the amount of cell damage 1 Electromagnetic radiation radio waves microwaves visible light ultraviolet light x rays and gamma rays a Energy travels in waves has no mass b Wavelength is inversely proportional to the frequency 2 Ionizing radiation gamma rays xrays a Destroys DNA and maybe the membrane b Produces reactive oxygen species that damage other cell components c Used to sterilize heatsensitive medical equipment disposable surgical supplies drugs spices herbs meat 3 Ultraviolet radiation a Damages DNA poor penetration b Used to kill microbes in the air on surfaces and in drinking water c Limitations i Cannot kill microbes inside solids or turbid liquids ii Most glass and plastic block UV light 4 Microwaves a Don t kill directly but by generating heat b Microwave ovens heat food unevenly so cells often survive c High pressure without heat i Destroys microbes by denaturing proteins and altering the cell s permeability 1 Used with some commercial foods guacamole 2 Avoids problems with high temperature pasteurization 3 Employs high pressure 4 Products maintain colour and avour 11 7 55 Using Chemicals to Destroy Microorganisms and Viruses a Useful for large surfaces and heat sensitive items but are less reliable b Target proteins DNA membranes or viral envelopes c Many are poorly understood d Potency of germicidal chemical formulations i Sterilants destroy all microbes used on heat sensitive critical instruments ii Highlevel disinfectants destroy all viruses and vegetative cells but do not reliably kill endospores used on semicritical instruments iii Intermediatelevel disinfectants destroy vegetative bacteria mycobacteria fungi and most viruses used on noncritical instruments iv Lowlevel disinfectants destroy fungi vegetative bacteria and enveloped viruses do not kill endospores or naked viruses used to disinfect furniture oors and walls e Important to consider i Toxicity bene ts must be weighed against risk of use ii Activity in presence of organic material many germicides react with organic matter losing effectiveness iii Compatibility with material being treated eg electrical equipment needs gas and not liquid that could damage metals iv Residues can be toxic or corrosive must be removed after treatment v Cost and availability eg bleach is cheap ethylene oxide isn t vi Storage and stability concentrated stock reduces storage space vii Environmental risk agent may need to be neutralized before disposal 12 Name Pros Cons Uses Examples Antiseptics Rapid prior to Ethanol Alcohols Cheap evaporation breaking isopropanol available limits skin rubbing contact time treatment of alcohol instruments Irritating to Sterilize Destroys all respiratory medical Glutaraldehy Aldehydes microbes tract skin equipment de fast and eyes preserve formaldehyd effective specimens e Low toxicity destroys Antiseptic in range of Rare but soaps and Biguanides microbes severe lotions in Chlorohexidi stays on allergic catheters ne skin and reactions and surgical mucous mesh membranes Penetrates Toxic doesn t explosive Sterilize Ethylene damage carcinogenic medical oxide gas moisture expensive devices sensitive not available Reacts with Chlorine organics to disinfects Inexpensive become objects and Chlorine and Halogens readily toxic or water iodine available inactive iodine is doesn t kill disinfectant endospores and anUsepUc Antimicrobia Too toxic for Dressing for l inactivates medical burns Metals proteins and settings prevent eye Silver enzymes pollutes infections in water strict newborns control industry used Oxidizing Chlorine agent Decompose alternative Ozone 03 decomposes s quickly water Ozone fast expensive disinfectant 13 Oxidizer Effective on Inanimate Peroxygens sterilant some objects Hydrogen biodegradab surfaces food peroxide e only industry 14 Nontoxic Inanimate react with Not objects Benzalkoniu Quaternary membranes effective on disinfect m cholride ammonium destroy endospores food compounds vegetative mycobacteri preparation Quats bacteria and a naked surfaces enveloped viruses health care viruses products Low cost Irritates destroys skin leaves Phenolics membranes residue not Used in earliest denatures reliable on soaps Triclosan disinfectant proteins all viruses lotions kills most can cause toothpaste vegetative neurological bacteria damage 1 Resistance and toxicity i Excessive use raises concerns 1 Disinfectants and antiseptics can provide selective pressure to acquire ef ux pumps which can expel a range of chemicals including antibiotic drugs acquired resistance 2 Toxicity affects microbiota allergic reactions to chemical disinfectants are a greater concern 3 Important to weigh the bene ts and risks of disinfectants and antiseptics 8 Preservation of Perishable Products a Prevent or slow growth of microbes to extend the shelf life Useful in production of food medicine deodorants cosmetics contact solution i Chemical preservatives 1 Food preservatives must be nontoxic 2 Weak organic acids 3 Nitrate and nitrite a lnhibit metabolism and alter cell membrane b Control molds and bacteria in foods and cosmetics a Processed meats hot dogs lunch meat b lnhibit endospore germination and vegetative cell growth 15 c Higher concentrations give meats pink colour can form carcinogenic compounds during cooking ii Lowtemperature storage 1 2 3 4 Refrigeration inhibits or slows growth by slowing or stopping critical enzyme functions Psychrotropic and psychrophilic organisms can grow at this temperature Freezing preserves by stopping all microbial growth Some microbial cells are killed by ice crystal formation but many survive and grow once thawed iii Reducing water availability 1 2 3 Sugar and salts draw water out of cells osmosis dehydrating them Halotolerant microbes can survive high salt concentrations Drying foods lyophilization freezedrying a Compiled with addition of sugar andor salt b lnvolves freezing then drying under a vacuum c Used for milk meat produce can be rehydrated d Does not reliably kill microbes only stops growth 16 Chapter 7 Bacterial Gene Regulation Victoria Gonzalez Terms Quorum sensing communication between bacterial cells by means of small molecules permitting the cells to sense the density of cell Twocomponent regulation mechanism of gene regulation that uses a sensor and a response regulator Response regulator the protein that binds to DNA in a two component regulatory system Repressor protein that binds to the operator site and prevents transcription Operator region located immediately downstream of a promoter to which a repressor can bind binding of repressor to the operator prevents RNA polymerase from progressing past that region thereby blocking transcription lnducer substance that activates transcription of certain genes Corepressor molecule that binds to an inactive repressor thereby allowing it to function as a repressor Monocistronic RNA transcript that carries one gene Polysistronic an mRNA molecule that carries the information for more than one gene Operon group of linked genes whose expression is controlled as a single unit Regulon set of related genes transcribed as separate units but controlled by the same regulatory protein Sigma factors component of RNA polymerase that recognizes and binds to promoters CAP cataboliteactivating protein activator involved in carbon catabolite repression Lac operon operon that encodes the proteins required for the degradation of lactose serves as one of the most important models for studying gene regulation Key concepts How prokaryotic and eukaryotic gene expression differ How bacteria sense changes in the environment Difference between constitutive inducible and repressible operons Mechanisms for bacterial gene regulation transcriptional translational posttranslational 0 Alternative sigma factors 17 o DNAbinding proteins 0 How Ecoi lac operon is regulated How to make predications about gene expression from an operon model 1 74 Differences Between Eukaryotic and Prokaryotic Gene Expression a Location and timing i Prokaryotes transcription and translation are coupled ii Eukaryotes they occur in separate locations in the cell b Processing i Prokaryotes processing does not occur ii Eukaryotes premRNA is processed to mature mRNA by the addition of a 5 cap and a 3 polyA tail c Genes per transcript i Prokaryotes mRNA is polycistronic ii Eukaryotes mRNA is monocistronic d lntrons and splicing i Prokaryotes mRNA does not contain introns ii Eukaryotes mRNA contains introns they are removed via splicing 2 75 Sensing and Responding to Environmental Fluctuations a Bacterial gene regulation i Microorganisms constantly face changing environments 1 Nutrient availability temperature cell density etc ii Must be able to adapt quickly in order to survive iii Signal transduction transmits information from outside the cell to inside b Bacterial sensing i Quorum sensing chemical signaling between cells 1 When there is few cells low concentration of signaling molecules 2 When there are many cells high concentration of signaling molecules that induce changes in gene expression 3 Ex bio lm creation is only expressed when there is a high density of bacteria ii Twocomponent regulation signal transduction 18 Bacteria cells are able to sense and respond to changes in their environment Sensor protein detects changes in the environment Response regulator initiates changes in gene expression turns genes on or off 19 3 76 Bacterial Gene Regulation a Operon genes transcribed as a polycistronic message i Grouping quick and ef cient regulation ii Lac operon transports and hydrolyzes lactose b Regulon network of genes or operons controlled by a common regulatory mechanism i Global simultaneous control c 3 types of genes based on regulation i Constitutive always expressed quotonquot 1 Typically have indispensable roles in central metabolism ii Inducible not routinely expressed 1 Only turned on when needed 2 Avoids waste of resources 3 Ex transport and breakdown of alternative energy sources iii Repressible routinely expressed 1 Turned off when not needed 2 Allows cells to conserve resources 3 Ex biosynthesis of amino acids d Most genes need to be expressed at different times and levels e Sigma factors loose component of RNA polymerase that recognizes speci c promoters i Standard sigma factor recognizes most promoters for normal growth conditions f 2 mechanisms are used to control transcription they are readily reversible to allow cells to control the relative number of transcripts made i Alternative sigma factors recognize different sets of promoters to control the expression of speci c groups of genes ii DNAbinding proteins 1 Repressors block transcription negative regulation a They bind to operator sequences and block RNA polymerase movement or binding b Induction an inducer is used to prevent repressor binding c Repression a corepressor can be used to promote repressor binding 2 Activators promote transcription positive regulation 20 a They bind to a site near the promoter and often directly interact with RNA polymerase b Induction inducer is used to promote activator binding NAainding Prteins Reireaswra I ilmquot imi minhmwm swamaim mnmjm h fw ll l i l m Tmmldwwlm i w memmmimwmwmmam 2 1 NAginadmg Pmteins Activat rs iv i n i Il ndwer 7 PE l 39 f Fun lu al Acihmtm A tl wtair Trincrilp nn39 H iU d atwa m mmme th nJymeaEEammier kindling 22 g Lac operon vi vii viii Genes are needed for lactose utilization lactose is fuel Best studied in Ecoi Uses repression and activation Lactose 1 No lactose repressor prevents transcription by binding to operator sequence on the DNA turns lac genes off 2 Lactose present converted to allolactose inducer which binds to the repressor and prevents DNA binding transcription occurs lac genes are on a Transcription of lactose operon occurs only when glucose levels are low glucose is preferred energy lnducer exclusion lactose transporter is blocked during glucose transport Catabolite activator protein CAP required for transcription cAMP ATP derivative Glucose 1 No glucose cAMP levels rise CAP binds and transcription is activated lac genes are on 2 Glucose present cAMP levels are low CAP cannot bind lac genes are off 23 Namia ijn MTWATE Law transm p m Tram ma Ilai QIMEEEE ram Mlulia me hilmls ln the pxrmm ilwg Ei El39aitEiI39 Ila irndlng hanmrlptmm E l m l Ei d M Ih p i xl vinhih L31 quot ll a m e L V transcripti n U 1 lath rangiulal pmtelnz I and mesh 24 Chapter 8 Bacterial Genetics Victoria Gonzalez Terms Genotype the sequence of nucleotides in an organism s DNA Phenotype the observed characteristics of a cell Wild type form of the cell or gene as it typically occurs in nature Auxotroph a microorganism that requires an organic growth factor Prototroph a microorganism that does not require organic growth factors Point mutation mutation in which only a single base pair is altered Silent mutation a mutation that does not change the amino acid or phenotype encoded Missense mutation mutation that changes the amino acid encoded by DNA Nonsense mutation a mutation that generates a stop codon resulting in a shortened protein Nullknockout mutation gene is completely inactivated Transposon segment of DNA that can move from one site to another in a cell s genome Spontaneous mutation mutation that occurs naturally during the course of normal cell processes Induced mutation mutation that results from the organism being treated with an agent that alters its DNA Conjugation mechanism of horizontal gene transfer in which the donor cell physically contacts the recipient cell DNAmediated transformation mechanism of horizontal gene transfer in which the bacterial DNA is transferred as naked DNA Horizontal gene transfer DNA transfer from one bacterium to another by conjugation DNAmediated transformation or transduction Nonhomologous recombination DNA recombination that does not require extensive nucleotide sequence similarity in the stretches that recombine Plasmid an extrachromosomal DNA molecule that replicates independently of the chromosome Transduction mechanism of horizontal gene transfer in which bacterial DNA is transferred inside a phage coat 25 Key Concepts Differences between silent missense and nonsense mutations and how each might affect the coded protein s folding and funcUon What causes frameshift mutations and why they are so detrimental Transposons and how they might inactivate a gene Differences between spontaneous and induced mutations and examples of mutagen types Types of DNA repair mechanisms and the differences between them Three types of horizontal gene transfer transformation transduction and conjugation and a general idea of how each process words and the differences between them What will determine if a bacterial cell will pass on newly acquired DNA to its daughter cells What determines whether a mutation or newly acquired piece of DNA becomes dominant in a population Common examples of types of genes bacteria often pass around and maintain Difference between direct and indirect selection of mutants and when each are appropriate to use 81 Genetic Change in Bacteria Bacteria adapt to changing environments natural selection through a Regulating gene expression b Genetic changes Bacteria are haploid they have only one copy of a gene no backup copy A genetic change leads to a change in the genotype which may in turn lead to a change in the phenotype a Ex If a gene for tryptophan biosynthesis is disrupted the mutant will only grow is tryptophan is supplied in the growth medium i Mutant cell Trp auxotroph mutant that requires a growth factor increase nourishmentquot ii Wild type starting strain Trp prototroph does not require external tryptophan earliest form ofquot The phenotype of an organism can be altered not only by the genotype but by environmental conditions too Genetic change in bacteria occurs by 26 a Mutations change the existing nucleotide sequence of a cell s DNA which is then passed on to the progeny daughter cells through vertical gene transfer b Horizontal gene transfer acquisition of genes from another organism which is then passed on to the progeny 82 Spontaneous Mutations 1 Spontaneous mutation genetic changes that result from normal cell processes a Occur randomly and infrequent but at characteristic rates i Mutation rate probability of a mutation in each cell division 1 Between 10394 and 103912 for a given gene b Mutations may or may not be bene cial c Spontaneous mutations occur routinely so large populations contain mutants cells in a colony are not identical i Mutations are passed on to progeny ii Gives a population a chance to adapt to changing environments evolution iii The environment does not cause a mutation but rather selects for bene cial mutations iv Antibiotics select for resistant bacteria d Reversion mutation is occasionally changed back to the wild type 2 Two types of mutations a Base substitution mutations 1 Incorrect nucleotides are incorporated during DNA synthesis 2 Point mutation single base pair change 3 Outcomes a Silent mutation codes for the same amino acid as the wild type mutation does not affect the phenotype b Missense mutation codes for a different amino acid may or may not change the function c Nonsense mutation codes for a stop codon create for shorter and often non functional proteins 4 Knockout null mutation gene is inactivated 5 More common in aerobic environments 27 b Frameshift mutations additiondeletion i Effects depend on the number of nucleotides added or deleted 1 1 or 2 nucleotides results in frameshift mutation a Changes the reading frame of the mRNA b Different codons are translated c Protein is often shortened and nonfunctional knockout mutation 2 3 nucleotides one codon a Codes for 1 amino acid moreless b Impact depends on the location of the codon in the protein 28 4 Transposon mutations addition mutation a Transposons jumping genesquot i Can move from one location in the genome to another 1 Transposase enzymes move the DNA to a new location cut and pastequot 2 Often inactivates gene into which it inserts 3 Cannot replicate independently ii Antibiotic resistance genes can move this way b Barbara McClintock 19405 won Nobel Prize in 19805 i First evidence that genomes move ii Observed color variation in corn kernels resulting from transposons moving into and out of maize genes controlling pigment synthesis 83 Induced Mutations 1 Induced mutations genetic changes that occur due to an in uence outside of a cell such as exposure to a chemical or radiation 2 Mutagen an agent that induces change and increases mutation rate a Chemical mutagens chemicals that modify or resemble DNA bases often results in base pairing mistakes i Alkylating agents add alkyl group nucleotide substitution ii Nitrous acid HNOZ converts cytosine C to uracil U base pairs with adenine instead of guanine nucleotide substitution iii Base analogs resemble nucleobases but hydrogen bond differently nucleotide substitution iv Base substitutions are more common in aerobic environment 1 Reactive oxygen species ROS are produced from oxygen and can damage DNA 2 Increased levels of ROS are often produced by host cells as an antimicrobial defense mechanism they damage DNA proteins lipids etc b lntercalating agents insert between adjacent base pairs in the DNA strand this pushes the nucleotides apart space between base pairs cause errors in replication i Increase the frequency of frameshift mutations 29 ii If it intercalates into template strand a base pair will be added as the new strand is synthesized iii If it intercalates into the strand being synthesized a base pair will be deleted iv Examples 1 Ethidium bromide useful for staining DNA in the lab probable carcinogen 2 Chloroquine used to treat malaria 3 Radiation a Ultraviolet light UV induces thymine dimer formation i Covalent bonds form between adjacent thymine bases in DNA producing thymine dimers ii Thymine dimer distorts the helix which stalls replication and transcription iii Cell will die if the damage is not repaired Elil 1111 I I11 I 111 Igr Innu r e LlerI Ir iuh Ei hL s fwi whm b X rays i Cause single and double strand breaks in DNA 1 Double strand breaks result in deletions that are lethal to the cell ii Also alter nucleobases 84 Repair of Damaged DNA 1 Mutations are rare because DNA alterations are generally repaired before they can be passed on to progeny a First line of defense DNA polymerase proofreading i DNA polymerases synthesize DNA and proofread verify the accuracy of their actions ii DNA polymerases can back up and remove a nucleotide that is incorrectly bonded then incorporate the correct nucleotide b Second line of defense DNA repair mechanisms i Mismatch repair 1 A protein recognizes the mismatch 2 An enzyme cutes the sugarphosphate backbone near the sire of the mismatched base 30 An enzyme degrades a short stretch of the strand that had the error DNA polymerase synthesizes a new stretch incorporating the correct nucleotide DNA ligase glues the ends of newly synthesized segment to the original strand 31 Repair of modi ed nucleobases Glycolyase enzyme that removes an oxidized 1 2 is a g 5 33 4 3 5 ICTV iiiaria nucleobase At the site of the missing nuclobase an enzyme cuts the sugarphosphate backbone DNA polymerase degrades a short stretch of the strand DNA polymerase lls the gap and DNA ligase seals it W Elite lt h 4 Fill I Eu F 393 ewneine xi lful l guanine Gum ee e reeull el ramdeter damage Ehmeeyieee rernmies 1he emee nueleeEHse lrern the r eruirwrnpheeehebe meemim Al the erte all the miesing u l EII I 39IE I IEE39y39I39I rIE Eur 393 1m eargarraphueel reize baghglmne eel pelifrnerase degrades a ere ftEt 39i gr quotme Efrem the eem eme aerial5 e5 Driller EiIjlsirl iilrg zl39 g ig39 intl DEFm mama hill in entl eeell the gen Repair of thymine dimers UV light damage 1 Photoreactivation light repair light energy is used to break the covalent bonds of thymine dimers a Only used in prokaryotes 2 Excision repair dark repair enzymes recognize and repair major distortions in DNA 32 Ex39gilal repair Hr Mimi ITEIT EI El quot T i 39 ii u drum mums ElfM it lirtml 339 5w thtreaintiualiian ISL 7 m F 39r lifir lam 51 L E39 393 Jill Enzyme IiiPrimes Elm diirruig39az in I ivreualnirm m I I3 i t i lquoti w lquot iltii m E l j gl quot I a a T ii t 1 I f magmamm 635339quot ande t in r 3 LLiLIIIIiIl 39 manma thmm39 5 339 imammnmnnchmw t 39Ea39 3 35 WIMP iii ill HEM lagil39ii m I prim3m and Eh lgam quotHm 31 5 T T E 1 ihmnlilhn EEI39riI39ilj39 1ltlfafltlil1ll391n 3 1 LI Li i I l i v 1 i 5 m jr ai 39339 i535 3913 Jil J Equot lll39g39ll39ifl ElamEli tunquotsimian ll rL i llet iv SOS repair a global lastditch repair mechanism induced when the extensive DNA damage cannot be xed by photoreactivation or excision repair 1 Damage to DNA leads to stalling which is lethal 2 Has no proofreading ability errors made here are called SOS mutagenesis 85 Mutant Selection 1 Testing for phenotypesmutations a Direct selection positive selection used when mutants can grow in conditions in which parent cells cannot i Cells are inoculated onto a medium that supports the growth of the mutant but not the parent ii Ex an antibiotic medium is used cells that grow are antibiotic resistant mutants b Indirect selection negative selection used to isolate auxotroph from prototrophic parent a technique called replica plating is used i A plate of bacterial colonies is pressed onto the surface of sterile velvet ii Cells adhere to the velvet resulting in exact replicas of the original plate iii Two agar plates one nutrient agar and one glucose salts agar are pressed onto the velvet this makes replica plates iv The replica plates are incubated 33 v Prototrophs grow on both media but auxotrophs grow only on the nutrient agar 1 Because glucosesalts is a chemically de ned medium with no added growth factors 2 Auxotrophs need to be provided with growth factors I Amvummu m im I mlnw i 2 Ames test used to test if a substance is a mutagen or carcinogen Wilt l 3 Horizontal gene transfer when microorganisms acquire genes from other cells 34 Genes can be transferred from a donor to a recipient by three mechanisms a Transformation naked DNA uptake by bacteria b Transduction bacterial DNA transfer by bacteriophages c Conjugation direct DNA transfer between bacterial cells Recipient cells of gene transfer must replicate the new DNA to pass it on to daughter cells a Can only happen if the DNA is a replicon it contains its own origin of replication i Plasmids and chromosomes are replicons ii Fragments of chromosomal DNA are not replicons 35 Nl 86 Transformation Transformation uptake of naked DNA by recipient cells In order for transformation to occur the recipient ce must be competent in a speci c physiological state that allows the cell to take up DNA a The process of developing competence is tightly regulated i Some species are always competent ii Some species require a speci c density of nutrient conditions b Ex Bacillus subtiis has a twocomponent regulatory system i Recognizes low nitrogen and carbon supply and high concentration of bacteria to become competent Most bacterial cells take up DNA from any origin some accept DNA only from closely related bacteria Newly acquired DNA must recombine with the host chromosome to be maintained Process of transformation a Doublestranded DNA binds to speci c receptors on the surface of competent cells b One strand enters the cell the other strand is degraded i In the cell one strand is old sequence one is new sequence c Strand integrates into the recipient cell s genome by homologous recombination The strand replaced is degraded d Chromosome is replicated the cell divides and only one daughter cell inherits the donor DNA e DNA transformation is detected in labs by growing in selective conditions i On a strepcontaining medium StrS cells die but transformed StrR cells multiply selective pressure 6 Grif th s experiment transformation of pneumococci a Helped understand the structure and function of DNA b Pneumonia is caused if the pneumococci create a polysaccharide capsule 36 va quot 5 mmd39a 39 LIHing EnEaputla tE nzal IE 39i39 I f 1 5 39 Ha arrreact 1 39 A r 39 r 39 I D JIF39 l aquot I l Elwin munEmpaulaftad cells g 39 L A r v quot392 V 1 Liiwkn manmpaul ftad calla quot quot HE Iiliill Empaulaftad main WHEN l H e m I Li ngsanpaulatard 4h I lliii t d 37 87 Transduction 1 Transduction transfer of genes by bacteriophages bacterial viruses a Generalized transduction any genes of donor cell b Specialized transduction transfers speci c genes 2 Bacteriophage aka phage DNA or RNA covered in a protein coat a Phages infect bacteria by attaching to cell and injecting its nucleic acid b Enzymes made from phage genome cut bacterial DNA into pieces c Cell s enzymes replicate the phage s nucleic acid and makes more protein coats making new phages d Phages are released from the cell causing the cell to lyse e New phages infect other bacterial cells 3 Generalized transduction a Any gene can be transferred this way b Occurs due to a packaging error during phage assembly c Process i A bacteriophages attaches to a speci c receptor on a host cell ii Phage DNA enters the cell and the empty phage coat remains attached to the outside of the bacteria iii Enzymes encoded by the phage genome cut the bacterial DNA iv Protein coats and phage nucleic acids are synthesized v A fragment of bacterial DNA mistakenly enters the protein coats and creates a transducting particle bacterial DNA surrounded by a phage coat vi Transducting particle carries bacterial DNA to other cells inject DNA which may integrate via homologous recombination 4 Specialized transduction a Only bacterial genes adjacent to integrated phage DNA are transferred b Due to excision mistakes during induction of a temperate phage c Process i A temperate phage injects its DNA into bacterial host ii The phage DNA integrates into the host cell DNA to become a prophage 38 vi When the prophage is excised from the bacterial chromosome a mistake is made some bacterial DNA is integrated in the phage DNA and some phage DNA is left behind Replication and assembly of new phages produces defective phage particles that carry a certain bacterial DNA in place of some phage DNA The DNA of the defective phage is injected into the new host but cannot cause a productive infection The bacterial DNA integrates into the host genome via homologous recombination it can now be replicated alone with the rest of the host s DNA 39 88 Conjugation 1 Conjugation DNA transfer between bacterial cells a Requires contact between donor and recipient cells 2 Conjugative plasmids direct their own transfer from donor to recipient cells Two types of transfer plasmid transfer and chromosome transfer Plasmid transfer a Ex F plasmid of E coli is a conjugative plasmid it encodes proteins that promote transfer i The F pilus makes contact with the recipient F39 pilus ii The pilus retracts pulling the donor and recipient cells closer iii A single strand of the F plasmid is transferred to the recipient cell a complement is synthesized for each strand iv At the end both the donor and the recipient are F 5 Chromosomal transfer a Chromosomal transfer is less common than plasmid transfer b Involves Hfr cells high frequency of recombination cells i Cells in which the F plasmid has integrated into the chromosome through homologous recombination this process is irreversible ii Hfr cell produces F pilus c Process i The F pilus contacts the recipient F39 cell ii A single strand of the donor chromosome begins to be transferred starting at the origin of transfer 1 Gene A closest to the origin is transferred rst iii DNA synthesis creates complementary strands in both cells iv The donor and recipient cells separate interrupting DNA transfer v The donor DNA is integrated into the recipient cell s chromosome by homologous recombination Unincorporated DNA is degraded vi The recipient cell remains F39 00 40 89 The Mobile Gene Pool 1 Genomics reveals variation in the gene pool even within a single species a Only 75 of E coli genes are found in all strains b Core genome the conserved genes present throughout all strains in a species c Mobile gene pool genes that vary among different strain i Plasmids transposons genomic islands phage DNA ii Can move from one DNA molecule to another d Transposon segment of DNA that can move to different locations in the DNA of the same cell e Genomic islands large fragment of DNA codes for genes that allow cell to occupy speci c environmental locations f Phage DNA may encode proteins important to bacteria g Plasmid code for nonessential genetic information i Circular doublestranded DNA with an origin of replication 1 Replicate automatically 2 Low copy plasmids only onea few copies per cell high copy plasmids many copies in the cell 3 Can have narrow or broad host ranges ii Plasmids are found in most bacteria and archaea in some Eucarya iii Resistance plasmids encode resistance to different antibiotics 41 Khmer Gene Ie Chenuglee Mut H l i t l GEI IE TI HI TIE IEI Eeee Inee ienf eleiien Trene ermei en Tr nmu i n lu m Eiler Tre eeeeeee mem Fleem d er I Mieeenee EB mamv Liner eieeee Neeee ee r V 1 Fleemider Generelneee h Seeeielieed mmme IHGT new muei he neeed en te megeey infe ne ehremeeeme er have eel e gie in render e be Eenefie ehengee ml heeeme demieen in lime eemjletien i39lf they ere eeleete fer preside grmeih e e repre ueliee ede nriegeej 42 Chapter 13 Viruses Viroids and Prions Victoria Gonzalez Terms Bacteriophage phage a virus that infects bacteria Virion viral particle viral particle in its inert extracellular form Nucleocapsid viral nucleic acid and its protein coat Capsid the outer covering of a protein that protects the genetic material of a virus made up of capsomer subunits Burst size number of newly formed virus particles released when the infected host cell lyses Lysozyme enzyme that degrades the peptidoglycan layer of the bacterial cell wall Latent infection viral infection in which the viral genome is present but not active so viral particles are not being produced Lysogen a bacterium that carries phage DNA integrated into its genome Lysogenic conversion a change in the properties of a bacterium conferred by a prophage Lytic infection viral infection of a host cell with a subsequent production of more virus particles and lysis of the cell Productive infection viral infection in which more viral particles are produced CRISPR mechanism by which bacterial cells maintain a historical record of phage infections and thereby become immune to subsequent infections by the same phages the system also protects against other types of foreign DNA Restriction enzyme type of enzyme that recognizes and cleaves a speci c sequence of DNA Antigenic drift slight changes in a viral surface antigen that render antibodies made against the previous version only partially protective Replicase general term for any phageencoded enzyme that replicates the genome of an RNA phage Reverse transcriptase enzyme that synthesizes double stranded DNA complementary to an RNA template Plaque assay a method used to determine the number of viral particles in a suspension Prion infectious protein that causes a neurodegenerative disease Viroid an infectious agent of plants that consists only of RNA 43 44 Key Concepts 39RPUN Basic virus structure and how they care classi ed Differences between Iytic temperature and lamentous phages Generalized vs specialized transduction role in horizontal gene transfer The 5 step of virus infection general concepts needs a receptor can fuse or enter by endocytosis etc Compare and contrast genetics of DNA RNA and RT viruses do they need a replicase or transcriptase of their own Why some viruses mutate and evolve so quickly Acute vs persistent infections chronic vs latent How a virus infection can lead to cancer How viruses are cultivated in the lab and quanti ed Viroids and prions as infectious agents 131 General Characteristics of Viruses Viruses are obligate intracellular parasites a Not alive because they are incapable of metabolism replication or motility without a host cell b Infectious agents Viruses infect eukaryotic hosts Bacteriophages phages infect prokaryotic hosts Genetic information in a protein coat a Hijacks host cell s replication machinery b Directs host cell to make viral gene products Viruses are dif cult to study a Require live host cells b Most are unobservable with a light microscope Viruses are important to study a They cause disease b Bacteriophages are easier to study in the lab used as model systems i Vehicles for horizontal gene transfer ii Affect environmental bacteria populations kill bacteria Virus size a 1001000 times smaller than the cells they infect b Smallest 10 nm about ten genes c Largest 800 nm about 1000 genes 45 8 Viral architecture a Virion viral particle consists of nucleic acid surrounded by a protein coat i Nucleocapsid nucleic acid capsid ii Capsid protein coat protects nucleic acid from enzymes and toxic chemicals 1 Capsomers capsid subunits that determine the shape of a virus b Enveloped virus virus with lipid bilayer surrounding the outside of the capsid i Matrix protein between the nucleocapsid and the envelope ii Enveloped viruses are more susceptible to disinfectants because chemicals damage the envelope making the viruses noninfectious c Naked virus viruses that do not have an envelope i Almost all viruses d Spikes or tail bers on the outside help with host attachment e Nucleic acid genome i Can contain either DNA or RNA never both ii Can be linear circular or fragmented iii Can be double or single stranded Ell Fquot l39 IEI a fig r a H Ea i i 39l l Mania Elm Iiilir m subunils u I 4 39 7 n r Harrier acid WE 393 Wm V 39f g V llducleccapsid w Ea mj mmm H39 5 my ema EastStd l entre 7 H prefarr will jljl39 ij il39l Eriml Suit135 mail quot 7 7 E i l l i39iME f Three shapes of viruses i lcosahedral appears spherical but has 20 at faces requires least amount of energy to assemble ii Helical appear cylindrical capsomers arranged in a helix can be short or long and rigid or lamentous iii Complex icosahedral head and helical tail phages are complex viruses 46 9 Viral taxonomy controlled by International Committee on Viral Taxonomy a About 3186 species of viruses grew by 900 in 5 years b Key characteristics for classi cation i Genome structure 1 Is nucleic acid DNA or RNA 2 Is it double or single stranded ii Envelope or naked iii Hosts infected iv Shape v Disease symptoms vi Location c Viruses are grouped and informally named based on their route of transmission i Ex fecaloral route enteric respiratorysalivary route respiratory vector zoonotic sexual contact sexually transmitted 47 132 Bacteriophages 1 Phages are the most abundant and diverse genetic entity on the planet a 1031 phage particles exist globally b 1023 phage infections per second 2 3 types of phages based on host relationship a Lytic phage i Lytic or virulent phages exit the host by ysing the cell ii Host cell is ysed and dies iii Productive infection creates new phage particles iv EX T4 phage infects E coli v Five step process 1 Attachment a Phage particles collide with host cells by chance b Phage tail bers bind with bacterial receptors 2 Genome entry a Lysozyme enzyme on the tip of phage tail degrades a portion of the peptidoglycan in the bacterial cell wall b The phage s tail contracts and injects nucleic acid into the bacterial cell 3 Synthesis of proteins and genome a The cell begins synthesizing T4 phage proteins using hose ribosomes b Early proteins proteins that degrade host DNA proteins that modify host cell s RNA polymerase so it does not recognize bacterial promoters c Late proteins make structural proteins capsid tail etc 4 Assembly maturation a After phage components are produced they assemble to make new phages b Some components sef assemble spontaneously c Some require protein scaffolds 5 Release a Lysozyme is produced late in infection digests cell wall from within causing the cell to yse and release the new phages 48 b Burst size number of phage particles released i T4 burst size 200 phages cell vi Full process takes 30 minutes vii Generalized transduction packaging errors where the phage takes fragments of host DNA 49 b Temperate phage some phages enter lytic cycle some Iysogenize incorporate the phage DNA into bacterial DNA the cell i Lysogenic infection phage DNA is incorporated into the host genome 1 Phage remains latent no new phage particles are made at this point ii Lytic infection productive injection makes more copies of the phage DNA iii Lambda phage is a thoroughly studied temperate phage 1 Lambda DNA integrates into E coli host chromosome through site speci c recombination a Integrated phage DNA i Replicates with the host chromosome ii Excised by phageencoded enzyme Iytic infection b A transcription repressor protein phage encoded prevents expression of Iytic genes c Phage induction i DNA damage signals repressor cleavage ii Lytic genes are turned on iii Phage escapes damaged host Iytic c Filamentous phage i Single strand DNA phages that look like long laments ii Cause productive infections more viorns are produced iii Host cell is not killed by lysis it just grows slowly iv M13 phage is a well studied model 1 M13 attaches to F pili protein on E coli 2 Single stranded DNA genome 3 Host DNA polymerase synthesizes complementary DNA strand producing a double stranded DNA intermediate a One is the template for mRNA and the template for more genomic DNA b Other strand serves as the phage s genome itself 50 51 134 Bacterial Defenses Against Phages Preventing phage attachment a Bacteria can alter or cover its surface receptors to prevent infection b Sometimes covering surface receptors has other functions too c EX Staphylococcous aureus makes quotprotein Aquot that covers its phage receptors i Protein A also helps S aureus evade its host s immune system Restrictionmodi cation systems a Bacteria degrades incoming foreign DNA b Restriction enzymes recognize short sequences and cut foreign DNA i Bacteria s own DNA is methylated which the restriction enzymes don t recognize as foreign CRISPR clusters of regulatory interspace short palindromic repeats a Cells that survive phage infection store some of the phage s DNA sequence in their bacterial genome b Between each phage sequence is a repeat sequence the bacteria recognizes to help discriminate its own DNA from foreign DNA c The CRISPR array is transcribed into RNAs which bind to CAS proteins and degrade phage infection when infected again i A form of adaptive immunity 52 H H H Hi quot4 53 136 Animal Virus Replication 1 Animal virus replication is a vestep infection cycle 2 Attachment a Viruses bind to receptors usually glycoproteins on the plasma membrane of the eukaryotic host cell b Sometimes it must bind to various receptors for secure attachment c Speci c receptors are required limits the range of the virus i Some viruses can t infect some cells dogs cannot contract measles from humans 3 Penetration and uncoating fusion or endocytosis a Fusion i Spikes on virion attach to speci c host cell receptors ii Virion envelope fuses with plasma membrane iii Viral envelope remains part of plasma membrane iv Nucleic acid separates from capsid r a H 39m a 391 Ear elm nil trialwr lfu39EEF39MlcanE EFT532 Skiff I iiuijgiu i ucan ul Jl E h ng MM r uff Lemma n j i figs u I f ii39 area ii gr 5 j Fl Fug i w 111 wirljf figaim swim113 39 U 1 5 Eggfr jquot a rat1 quotIiZ fir ery ill EFW be mume Fowl b Endocytosis i Attachment to receptors triggers endocytosis ii Plasma membrane surrounds virion forming an endocytic vesicle iii Envelope of virion fuses with endosomal membrane iv Nucleic acid separates from capsid v Naked viruses lack a lipid envelope 50 they rely on endocytosis 54 c Synthesis i Production 1 Expression of viral genes to produce structural proteins and enzymes capsid proteins and enzymes for replication 2 Make multiple copies of viral genome ii Replication strategies can be divided based on the type of genome of the virus 1 Replication of DNA virus a Replication occurs in the nucleus b Host enzymes are used c Host machinery is used for genome replication and gene expression 2 Replication of RNA virus a Replication occurs in the cytoplasm b Viral encoded enzyme replicase is used to make the viral genome i Repicase laclt5 proofreading 50 mutations are frequent c Single stranded RNA viruses need to package replicase i indicates the RNA functions as a mRNA d Host machinery is used for gene expression 3 Replication of reverse transcribing virus a Viral encoded enzyme reverse transcriptase is used for DNA synthesis b Host machinery makes double stranded DNA that is integrated into host genome and isn t eliminated can be latent or productive c Host machinery is used for gene expression d Hepatitis B is a unique example of a double stranded DNA retrovirus that uses 55 an RNA intermediate for genome replication d Assembly i Protein capsid forms ii Genomes and necessary proteins are packaged 56 e Release i Most viruses are released via budding ii Viral proteins that will become envelope spikes insert into the host s plasma membrane iii Viral matrix protein coats the inside of the plasma membrane iv Nucleocapsid extrudes from host cell and becomes coated with lipid envelope with protein spikes v Some obtain the envelope from organelles like the Golgi vi Naked virus is released when host cell dies by apoptosis initiated by virus or host I HutJ w miv39sutl Liquot quotiirquot nF tszt CM Bali amt7r we 33 ii a i a 151mi rayn p39zirm and EMS3215 mi WESF REEF E guiltJquot r1 5115 m 1 igiqtl lliP I EIELELEVEN EELILHILJ Irii i39 p 39fn livir I a Hf m F wgf z mk quotis sulfa 39F I311 iti ii i fh 39quot l r i39miiit i WIS1E nut n ultlrtam 39IFH11 IIL1 wilrif 39 j 137 Categories of Animal Virus Infections 1 Virus evolution Viruses replicate producing many progeny Transmission between hosts Random mutations replicase does not proofread Reassortment and recombination i Between two viruses ii Between virus and host e Antigenic drift mutations in surface proteins recognized by the host immune system selective pressure up C70 57 3 Types of infections a Acute infections i Rapid onset ii Short duration b Persistent infections i Virus isn t cleared after initial infection ii Continues for years or for a lifetime may or may not have symptoms iii Some viruses exhibit both chronic and latent symptoms HIV iv Chronic infections continuous production of low levels of virus particles Hepatitis B v Latent infections viral genome remains silent in a host cell cannot be eliminated and can later be reactivated physiological changes immunosuppression cell trauma fever etc vi Herpes 1 Virus becomes latent in the sensory cranial nerve near the brain after initial infection 2 When the virus is activated it moves back along the sensory nerve to the face and causes cold sores c The immune system and host cell type have HUGE roles in the type of infection quotEVMl mini llll quotiiinal ghiilfn Ema 39 a s lain 39 Qu I q as J rlb l 1 I 1 r x n l 397 a l l l A quot l quot H A A F rain Winn 39L V f 1 Ln39iim39i Law Hiu u iw inflation Fl i jxifii i ls Elmaeiian Hem43 twin m 39Jr39el Eliai5 reg mags ilitit39fai 253753 gra quot2 Chin quot rmw clam G Tflquoti quot i i New 39a39urtlw whims Irrm39istcrme mm as gjiirl cal flare t39arsla39mrj Pt r13 1273 Er39ri Jeffmquot 39a iquot ifu tcsi BEE flaws LT adding in 39rEH39 TECH htahtrra39 water quot 9 3 iii ti 39139 ii 1 39 tn g my 31351 58 59 138 Viruses and Human Tumors 1 A tumor is abnormal cell growth a Protooncogenes stimulate growth and cell division b Tumor suppressor genes inhibit growth and cell division c Viral oncogenes can interfere with host control mechanisms stimulating cell growth and inducing tumors 139 Cultivating and Quantitating Animal Viruses 1 Viruses must be grown in appropriate host cells a Primary cultures obtaining animal cells by removing tissue from an animal i Tumor cells are often used because they multiply inde nitely b Cytopathic effect changes in host cells due to a virus 2 Quantitating animal viruses a Plaque assays precise method for determining concentration of animal viruses in a sample by using monolayer of tissue culture cells b Direct counts used when there are high concentrations seen through an electron microscope c Quantal assay dilution that yields ID50 50 of cells are infected or LD50 50 of cells are killed d Hemagglutination when ce viruses cause red blood cells to clump can be used to determine relative concentrations 3 To study bacteriophages must note that viruses multiply inside living cells a Cultivate in suitable bacterial host cell b Plaque assays are used to quantitate phage particles in samples sewage seawater soil c Counting plaque forming units PFU yields titer 60 1311 Other Infectious Agents Viroids and Prions 1 Viroids small singlestranded circular RNAs a Only found in plants b Cause disease 2 Prions protein infectious agents a Linked to slow fatal human and animal disease neural tissue i Mad cow b Usually speciesspeci c transmission c Transmissible spongiform encephalopathy all prion diseases i Bovine spongiform encephalopathy mad cow disease d Process i Cells produce normal form of the protein ii Infectious prion protein is insoluble aggregate resistant to proteases heat and chemicals iii Infectious prion protein converts normal protein through a change in protein folds
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