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BIOL 440 > Exam 3 > Flashcards

Exam 3 Flashcards

1
Q

Define biotechnology. What are its applications?

A

use of cells and biological molecules to solve problems/make useful products

  • can eliminate undesirable phenotypic traits
  • combine beneficial traits btw 2 organisms
  • create organisms that synthesis products for human needs
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2
Q

What is recombinant DNA? What is its role in genetic engineering?

A

Recombinant DNA - process of cutting and combining DNA that contains genetic info from two different species of organism
- regular plasmid with newly introduced DNA strand
Genetic engineering - manipulation of an organism’s genetic info by introducing specific genes
- use recombinant DNA as a tool in genetic engineering

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3
Q

How would you get a pure culture of bacteria with a recombinant plasmid? What is a vector?

A

add R gene to plasmid - plate on antibacterial plate with specific R antibiotic and will only get cells with recombinant DNA to grow
- ex of a vector - NA molecules used to deliver a gene, typically with specific phenotype

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4
Q

Describe the process of reverse transcriptase. What are the benefits of this kind of NA synthesis?

A

uses RNA template to transcribe DNA for desired gene - create complementary DNA (cDNA)

  • easier to make mRNA for desired gene than trying to find and replicate specific gene on DNA
  • can be used to insert eukaryotic genes into prokaryotic organisms
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5
Q

What are restriction enzymes and what are the different types?

A

enzymes that recognizes specific DNA sequence and cuts DNA internally on both strands

  • staggered - at palindromes - creates “sticky ends” that allow for another piece of DNA with complementary DNA to bind
  • blunt - cuts straight down - more difficult because there it is not sticky, but are not specific
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6
Q

Describe the process of recombining DNA

A

restriction enzyme recognizes and cuts DNA internally on each strand
- staggered or blunt
DNA ligase is used to join new segment of DNA and forms permanent seal - forming recombinant DNA

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7
Q

What are some examples of recombinant DNA?

A

rBGH - growth hormone for cows - increases hormone - cows get shot to produce more milk
argobacteirum tumefaciens - plant pathogen
- bacteria injects pathogenic DNA into plant cell and produces crown gall

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8
Q

What is DNA profiling/fingerprinting? What are some examples?

A

analysis of fragments prepared from the DNA of an individual that can be used to distingue that individual from another

  • uses gel electrophoresis to separate molecules based on size, charge and shape
  • DNA - negative charge - smaller DNA travels faster and farther down gel
    ex. restriction fragment length polymorphism (RFLP) and PCR - polymerase chain reaction
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9
Q

Describe RFLP analysis.

A

restriction fragment length polymorphism/southern blot
uses restriction enzymes to cut DNA and analyze fragments
- fragments move down gel electrophoresis
- placed on nitrocellulose membranes - less delicate
- probes for DNA of interest - radioactive and complementary to DNA of interest - hit with radiation to reveal presence

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10
Q

What are the steps of PCR?

A
  1. denature - 92 to break H bonds and separate strands
  2. priming - mix excess of primers, nucleotides, DNA polymerase so it bonds to primers instead of strand
    - primers provide 3’ hydroxyl needed for DNA synth
  3. extension - 72 to increase rate of DNA polymerase
  4. repeated for exponential growth - thermocycler
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11
Q

Define genomic and bioinformatics. What are some possible microbial applications with microbial genomics?

A

genomics - study and analysis of nucleotide sequence of an organism
bioinformatics - use of computer technology to analyze biological information (sequence data)
potential - new therapies, preventative agents, renewable energy sources, clean up of toxic waste

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12
Q

Compare the following: aseptic, - static, and -cidal

A

aseptic - environment/procedure that is completely free of contamination by pathogens

  • static - inhibition of microbial growth
  • cidal - destruction of microbe
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13
Q

What are the differences between sterilization and disinfection?

A

sterilization - removal/destruction of all microorganisms
- not always possible i.e. beds, scopes
- practical terms - eradication of all harmful organisms
disinfection - reducing # of pathogenic microbes to point of no danger/disease
- antisepsis - use of disinfectant nontoxic enough for skin use i.e. iodine
- sanitation - disinfection to reduce pathogenic microbes to public health standards i.e steam in restaurants

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14
Q

What are the criteria for selecting a microbial control procedure?

A
  1. Where is the site?
    - external - typically not sterilized
    - internal - penetration = sterilization
  2. What is the susceptibility of microbes?
    - resistant microbes like endospores, mycobacteria, protozoan cysts and prions require special care
  3. What are the environmental conditions?
    - temperature - disinfectants work better in warmer temps
    - acidic conditions enhance antimicrobial effect
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15
Q

What are some ways to control microbial growth?

A

damage cell walls and membranes - cell lysis/death
damage to proteins - important for metabolism regulation, enzymes and structural proteins - cell - denatured - death
damage to nucleic acids - fatal mutations/ribozyme damage - no protein

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16
Q

What is the disk method and how is it used to measure microbial control?

A

Kirby-Bauer method
- spread bacteria evenly on agar, apply filter disks with microbial control agent
zone of inhibition - area of no bacterial growth around disks measured by diameter of clear region in mm
- larger the zone indicated more inhibition of bacteria
- size of zone must be compared to standard for particular drug

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17
Q

What is the decimal reduction time and what does it measure?

A

rate of microbial death

  • the amount of time required for a 10 fold reduction (90%) of microorganism death at a given temperature
  • log decrease by 1
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17
Q

What is the decimal reduction time and what does it measure?

A

rate of microbial death

  • the amount of time required for a 10 fold reduction (90%) of microorganism death at a given temperature
  • log decrease by 1
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18
Q

What are some heat related method of controlling microbial growth?

A

MOIST
Autoclave - sterilizing by moist heat - increasing T of steam by increase P
- 121 degrees at 15 lb/sq
Pasteurization - reduces microbial populations in heat sensitive foods
- flash - milk - 72 for 15 seconds
- ultra high pasteurization - 134 for 1 second
Ultrahigh temp sterilization - flash heat at 140 for 1-3 liquids - can be stored at room temp

DRY
flaming/incineration - ultimate means of sterilization
baking - takes longer for dry heat to penetrate

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19
Q

Besides heat, what are some other physical ways to control microbial growth?

A

refrigeration and freezing - decreases metabolism/reproduction of mesophiles
desiccation - drying - inhibits metabolism bc H2O required
lyophyilization - freezing and vacuum drying - preserves for years
filtration - through pores too small for microbes - sterilizes liquid

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20
Q

What are the kinds of radiation used for microbial control?

A

Gamma - ionizing radiation - dislodges e- from atoms
- produces radicals that react with DNA - denature DNA and cause cell death
- used in sterilization of medical supplies and food - expensive
- food - kills microbes, larvae/eggs, and prevents over ripening
UV radiation - nonionizing radiation
- DNA thymine dimers - prevents transcription/replication
- germicidal lamps - sterilizes surfaces

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21
Q

How do alcohols, aldehydes, and phenols/phenolics control microbial growth? What kind of method are they considered?

A

Chemical method
alcohol - denature proteins and damage membranes - not effective against spores
- tincture - mix of antimicrobial and alcohol
aldehyde - inactivate proteins and nucleic acids by cross linking
phenols/phenolics - disrupt membranes and denature proteins - effective in presence of organic material - lysol

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22
Q

How do halogens, oxidizing agent and heavy metal compounds control microbial growth? What kind of microbial control was?

A

Chemical microbial control
halogens - damage to enzymes by oxidation or covalent bonding ex. Cl, Br, Fl, I
oxidizing agent - oxidizes enzymes - peroxides, ozone and per acetic acid
heavy metal compounds - inactivates enzymes and other proteins - silver nitrate and thimersal (vaccines)

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23
Q

What are surfactants?

A

“surface active” chemicals - soaps and detergents

  • contain Quaternary Ammonium Compounds (Quats) that are cationic detergents
  • disrupts cell membranes
  • has hydrophobic ties that dissolve oil and associated bacteria to be washed away
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24
Q

describe the difference between bactericidal and bacteriostatic agents

A

bactericidal - agents that kill bacteria
bacteriostatic - agents that inhibit growth but do not kill bacteria
- gives immune system time to catch up

25
Q

What are antibiotic drugs? What are some types?

A

chemical agent produced by microbe that inhibit/kill other microbes

  • synthetic - produces in laboratory
  • antibiotic - produces by bacteria/fungi
  • semisynthetic drug - chemically altered form of antibiotic - can be made to be more effective, stable, easier to administer
26
Q

Describe the properties of antibiotics

A

Selective toxicity - harms the microbes without damaging the host
Spectrum of activity:
- broad - tetracycline kills Gram - and +
- narrow - isoniazid kills only mycobacteria
mode of action

27
Q

Describe drug synergism and antagonism

A

Synergistic - drugs given simultaneously greater together than given alone
- trimethoprim and sulfa
antagonistic - two drugs interfere with each other when given together
- PCN and tetra
additive - drug combination that is neither synergistic nor antagonistic

28
Q

How do beta lactams work?

A

Inhibits cell wall synthesis - causes cell to lyse
beta lactam drugs - PCN
- prevents cross-linking of peptidoglycan
- PCN effective against Gram +
- Ampicillin effective against G + and some -

29
Q

What are some antibiotic that inhibit cell wall synthesis?

A

bacitracin - topical, effective against G +
- blocks transport of NAG and NAM from cytoplasm

vancomycin - effective against G+

  • block Ala-Ala bridge btw NAM
  • effective against MRSA - toxic to humans

Isoniazid (INH) - narrow spectrum, synthetic
- inhibits mycolic acid synthesis

30
Q

What is the mode of antibiotic resistance related to beta lactams?

A

microbes have developed penicillinase (beta-lactamases) that cleave the beta lactam ring of PCN

  • developed new penicillinase drugs - methicillin and monobactam
  • developed penicillinase inhibitors - form of synergism: amoxicillin and clavulanic acid (non competitive inhibitor of penicillinase)
31
Q

What are some antibiotics that disrupt the cytoplasmic membrane of bacteria?

A

Polymyxin B - topical - effective against G-
- binds to cell membrane and disrupts structure and permeability

Polyenes (amphocerin B) - binds to sterols (ergesterol) in plasma membrane
- disrupts fungal plasma membrane by creating hole in membrane - lyses

Azoles (micon/clotrim) - systemic and topical
- inhibit ergosterol synthesis in fungi

32
Q

What are some antibiotics that inhibit protein synthesis?

A

Aminoglycosides - cause change in shape of 30s, mRNA is misread and wrong AA’s added
- ex. streptomycin, kanamycin, and neomycin

Tetracyclines - block tRNA docking site A and prevents incorporation of additional AA’s

Chloramphenicol - blocks enzymatic site of 50s, prevents formation of peptide bonds btw AA’s

Macrolides - binds to 50s subunit & blocks mRNA movement through ribosome - synthesis stops
ex. azithromycin and erythromycin

33
Q

What are some examples of antibiotics that inhibit nucleic acid synthesis?

A

Rifampin - binds to/inhibits RNA polymerase
- used to treat mycobacterial diseases

Quinolones - inhibits DNA gyrase - important for coiling/uncoiling for replication and prevents DNA replication
- Cipro

Nucleotide analogs - incorporates itself into nucleic acids to prevent further synthesis

  • distorts NA shape
  • developed from small changes to nucleoside
  • Acyclovir
34
Q

What are some antibiotics that inhibit metabolic pathways?

A

Sulfas
- sulfanilamide - analog of PABA that blocks enzyme that converts PABA into dihydrofolic acid

Trimethoprim - blocks enzyme at later step

  • prevents conversion of dihydrofolic acid into tetrahydrofolic acid
  • does not affect human metabolism bc we do not synthesize this from PABA
35
Q

What are some drugs that are used to inhibit pathogen-host interaction?

A

Fuzeon - prevents entry of HIV into host cells
- viruses attach to host cells via chemical interaction btw attachment proteins on pathogen and complementary receptor protein on host cells - works by preventing attachment and entrance

36
Q

What are the dilution methods of measuring antimicrobial activity? What is MIC and MBC?

A

Prepare a dilution series of an antibiotic and add a defined amount of microbes to each dilution

Minimum inhibitory concentration - lowest [drug] that inhibits bacterial GROWTH

Minimum bactericidal concentration - lowest [drug] that kills bacteria

37
Q

Describe the process of calculating MIC

A

broth dilution with increasing [antibiotic]
- each tube has same [bacteria]
- compare turbidity with spectrometry
lack of turbidity indicates bacteria inhibited by antibacterial agent
- want the lowest [drug] with no turbidity
- good for medications - want lowest [drug] in blood

38
Q

Describe the process of calculating MBC

A

broth dilution with increasing [drug] but same [bacteria]

  • get clear tubes and do a subculture by pouring and inoculating onto plate
  • clear MIC tube - can still have live bacteria and produce colonies on plate
  • MCB - lowest [drug] where subculture has no bacterial growth
39
Q

What are the clinical factors that are considered when choosing an antibiotic?

A
  1. spectrum of action
    - broad - effective against many different kinds of bacteria, can cause secondary infection
    - narrow - effective against only a few
  2. efficacy - disk diffusion, MIC, MBC
  3. routes of administration - oral, IM, IV
  4. safety and side effects - toxicity, allergies
    - disruption of normal microbiota: broad antiB decreases normal microbes and reduces microbial antagonism - natural competition
40
Q

What is the therapeutic index and window for a drug?

A

Index - ratio comparing largest dose of drug that isn’t toxic and lowest effective dose
window - range of [drug] that are effect without being excessively toxic

41
Q

What is antibiotic resistance and how does it affect the bacteria?

A

acquired ability of an organism to resist the effects of an antibiotic
- in regular settings, R cells area usually less efficient bc they use extra energy to maintain R genes and proteins

42
Q

How is antibiotic resistance acquired?

A

spontaneous mutation - slowly add antiB to tube and one randomly becomes resistant

horizontal gene transfer - R plasmids

  • transformation, transduction, conjugation
  • can be resistant to more than one drug and can transfer multiple genes at once

presence of antimicrobial agent does not produce resistance but selects for resistant microbes already presenting population

43
Q

What are some of the mechanisms of antibiotic resistance?

A
  1. drug inactivating enzymes (ex. B lactamases)
  2. alteration of target molecule - drug can no longer bind
  3. decrease entry of drug into cell (porins in Gram - )
    - changes in structure or electrical charges of membrane protein channels
  4. increased elimination of drug
    - through resistance pump - pushes drug out before it can be effective
  5. alteration of metabolic chemistry
    - producing more enzyme to increase pathway and antiB cannot keep up
44
Q

Describe a virus. What kind of nucleic acid does it have?

A

very small (10-300nm) infectious agent that replicates by invading cells - incapable to metabolic activity on their own
consists of nucleic acid surrounding by capsid
- can be single or double stranded DNA or RNA, but never both
- genome significantly smaller than cells
- can have multiple linear strands or single circular plasmid

45
Q

Describe the differences in virus states when it is extracellular and when it is intracellular.

A

Extracellular - virion - capsid surrounding nucleic acid
- can have an envelope

Intracellular - capsid and envelop are removed
- viral genome released into the cell and viral replication occurs

46
Q

describe the structure of the virus

A

Capsid - protein (capsomeres) coat surrounding nucleic acid

  • Nucleocapsid - capsid + nucleic acid
  • more stable outside host

Envelope - lipid bilayer and glycoproteins surrounding nucleocapsid

  • belongs to host - takes part of cell membrane
  • more stable inside host
  • no cytoplasmic membrane, cytosol, functional organelles

both acts as protection and recognition site that binds to complementary structures of functional organelles

47
Q

What are the virus morphologies? How are they classified?

A

helical - plants

polyhedral - humans

complex/head and tail - bacteria

viruses classified by nucleic acid, presence of envelope, and shape and size

48
Q

What are the steps in the bacteriophage lytic replication cycle ?

A

Viruses cannot reproduces themselves - all enzymes and organelles are hosts

  1. attachment - tail fibers with high specificity interacts with host cell receptors
  2. Entry - injection of nucleic acid into cell
  3. biosynthesis of nucleic acids and proteins
    - new copies of viral genome
    - proteins needed for virus coat
  4. assembly - construct complete iron from proteins and nucleic acid
    - spontaneous process - does not require enzymatic activity
    - can accidentally form capsid around hose DNA and inject into other host cells - transduction
  5. release - cell lysis and release of bacteriophage in lytic replication cycle
    - phage produces lysozymes
48
Q

What are the steps in bacteriophage replication?

A

Viruses cannot reproduces themselves - all enzymes and organelles are hosts

  1. attachment - tail fibers with high specificity interacts with host cell receptors
  2. Entry - injection of nucleic acid into cell
  3. biosynthesis of nucleic acids and proteins
    - new copies of viral genome
    - proteins needed for virus coat
  4. assembly - construct complete iron from proteins and nucleic acid
    - spontaneous process - does not require enzymatic activity
    - can accidentally form capsid around hose DNA and inject into other host cells - transduction
49
Q

What are the steps in lysogenic bacteriophage replication?

A

attachment and entry are the same, but cell does not lyse immediately

  • prophage/provirus - incorporates viral DNA into cell, cell replicates so both daughter cells have prophage
  • inactive virus - codes for a protein that suppresses prophage genes

induction - prophage get excised from chromosome and can reenter lytic phase

50
Q

What are the virus lifestyles?

A

virulent - viruses that kill the cells they infect

temperate - can undergo lysogeny

  • virus exists as prophage, viral genes turned off, replicated along with host genome
  • temperate viruses can undergo lytic growth after induction
51
Q

How are animal viruses classified?

A

based on nucleic acid type, virus shape, and presence of envelope

  • classified into families, no species
  • each family divided into genera
  • also known by common name
52
Q

What are the steps in animal virus replication?

A
  1. attachment - high specificity in interaction between host and glycolysis proteins in host cell membrane
    ex. HIV binds to CD4 receptor of T lymphocytes
  2. entry - 3 types
    - direct penetration - virion ejects genetic material directly into cell i.e. polio
    - membrane fusion - viral envelope and cell membrane fuse to allow entry of nucleocapsid into cell ex. measles and mumps
    - endocytosis - attachment to membrane followed by membrane surrounding virus - does not matter if it is enveloped or not
    - if capsid enters cell, it goes through uncoating to release NA
  3. release
    lysis - host cell killed
    budding - formation of envelope around virus from any cellular membrane - glycoproteins get pushed through membrane
    - does not lyse host cells
    - persistent infection - host cell sheds virus slow and steady
53
Q

describe how the zika virus enters an animal cell

A
  1. attachment via E protein
  2. entry via endocytosis
  3. release via budding and exocytosis
54
Q

Describe viral latency. Can all viruses do it?

A

latency - process by which an animal virus remains dormant in a cell

  • lifelong occupation of hose that can be dormant for years without symptoms
  • incorporated into host DNA and can be activated when immune system is decreased
55
Q

How do we grow animal viruses?

A

cannot live on their own and must be cultures in suitable host cells

  • living animals
  • embryonated eggs
  • cell cultures
56
Q

describe how to perform a viral assay

A

plaque assay - assume each plaque corresponds to a single phage spreading and multiplying

  • mix virus with cells
  • spread on culture dish to distribute cells on surface
  • observe plaques
57
Q

What are prions? How do they cause disease?

A

proteinaceous infectious particles that consists only of protein - no nucleic acid
- produces spongiform encephalopathy: plaques/holes in brain tissue

consists of singe protein PrP found in normal cells, found in A helices

  • changes shape into pathogenic form - B pleated sheets
  • templating - prion PrP acts as temple to change and refold normal PrP
58
Q

What are some examples of prion diseases?

A

Bovine spongiform encephalopathy - mad cow disease

scrapie - sheep and goats become irritable and develop intense itching, lose coordination

Creutzfeld-Jacob disease (CJD) - fatal - loss of motor control, paralysis, dementia

Kuru - “shakes” - similar symptoms to CJD - Fore population in Papua New Guinea, long incubation period

59
Q

Does PrP diseases appear in all organisms?

A

Animal disease - affects brain
does not happen in all organisms due to nearby proteins/lipid rafts in membrane force shape back into alpha helix
- human PrP misfiles only if it contains Met at 129th AA - in 40% of humans

BIOL 440 (8 decks)

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