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64 Cards in this Set
- Front
- Back
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what does ELISA stand for?
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Enzyme-Linked ImmunoSorbant Assay
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What is the main mechanism employed by IMMUNOAFFINITY MICROSCOPY?
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attach a heavy metal particle to an antibody to localize specific targets, e.g. virus particles in plasmodesmata!
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What are the 2 ways antibodies are prepared?
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POLYCLONAL AB: uses an animals immune system to produce polyclonal immunoglobulin in a serum.
Monoclonal AB produced by HYBRIDOMAS derived from a single cell line. |
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Name the constituents of an antigen.
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-Usually proteins or carbohydrates
-Protein antigenic determinants: 6–10 AA’s -Antigens have many ANTIGENIC DETERMINANTS (the part of an antigen that is recognized by the immune system, specifically by antibodies) |
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What are ANTIGENIC DETERMINANTS?
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The part of an antigen that is recognized by the immune system, specifically by antibodies
-Protein antigenic determinants: 6–10 AA’s |
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What are HYBRIDOMAS?
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Special cells used to produce MONOCLONAL ANTIBODIES:
Fusion of mouse spleen cell and a mouse cancer cell:fusion of a specific antibody-producing SPLEEN cell with a MOUSE CANCER cell (myeloma) that is selected for its ability to grow in tissue culture and for an absence of antibody chain synthesis. Very specific, often too specific, may have to blend several together |
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Basic antibody structure?
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Y shaped, with:
2 heavy chains, 2 light chains and 2 antigen binding sites |
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How are POLYCLONAL ABs produced?
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produced by inoculation of a suitable mammal, such as a mouse, rabbit or goat. -antigen is injected into the mammal.
-immunoglobulins produced specific for the antigen. -This polyclonal immunoglobulin is purified from the mammal’s serum |
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What kind of cells are antibodies found in?
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B cells
antibody at surface- when triggered, secretes many more antibodies |
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2 ways to dispose of antibody aggregates in the body?
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phagocytic cells ingest antibody aggregates
or special proteins in blood kill antibody- coated foreign molecules |
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How are antibodies raised in animals?
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antigen A is injected over weeks with multiple doses, causing the animal to produce several different antibodies in its blood, which is later harvested.
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what are ELISA's?
example? |
a "wet-lab" type analytic biochemistry assay that uses one sub-type of heterogeneous, solid-phase enzyme immunoassay (EIA) to detect the presence of a substance in a liquid sample or wet sample
example: pregnancy test. |
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Indirect ELISA?
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Common type of ELISA
-Primary antibody attached to antigen -secondary antibody attaches to primary and an enzyme with a color changing substrate- Color change indicates the presence of an antigen *Enzymes are catalytic and amplify the signal whereas the antibody provides the specificity for the target molecule! |
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What is an immunodiffusion assay?
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antibodies are injected into holes in agar and diffuse through- if they come in contact with an antigen they form a visual barrier at the confluence
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Agglutination?
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Agglutination is the clumping of particles...relevant because antibodies cause agglutination of particles.
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RFLP stands for?
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Restriction fragment length polymorphisms
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steps in RFLP?
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• Isolating DNA
• Cutting with DNA “restriction enzymes” • Separating DNA fragments by size on gels |
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Purpose & significance of RFLPs?
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-A type of “genetic fingerprinting”
-Distinguish individuals by evaluating differences at the DNA level |
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What is the role of restriction enzymes?
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molecular scissors, usually recognizes palindromic sequence and digest/cuts and adds a methyl group
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which enzyme seals dna segments back together?
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DNA ligase
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PCR means?
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Polymerase Chain Reaction
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What is the purpose of PCR?
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Allows specific amplification of a
defined region of DNA/RNA |
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How is the target DNA segement identified in PCR?
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Specificity is determined by the short
“DNA primers” used to define the ends of the amplified DNA molecule |
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Special enzyme that allows PCR to be used?
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heat-stable DNA polymerase = Taq polymerase
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What are some important applications of PCR?
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(PCR: A way to amplify a specific region of DNA)
• Species or pathovar-specific primers will permit detection in mixed populations at low levels • RAPD’s = randomly amplified polymorphic DNA’s (a type of genetic fingerprint) -Can be coupled to the use of RFLP’s to identify organisms taxonomically, e.g. the Phytoplasmas. |
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What are RAPD's?
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• RAPD’s = randomly amplified polymorphic DNA’s
(a type of genetic fingerprint) segments of DNA that are amplified are random a semi-unique profile can be gleaned from a RAPD reaction. useful for biological comparison, "fingerprinting" |
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"Taq" comes from the name...?
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Thermos aquaticus (from yellowstone heat vents)
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How long is a primer?
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15-25 nucleatides long
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5 main types of resistance?
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constitutive (always on)
Induced (response to attach/ active) Cytoplasmic resistance Horizontal Resistance (multi-gene, minor or polygenic resistance) Vertical Resistance (single-gene, major, monogenic resistance) |
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Cytoplasmic resistance?
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normally mitochondia bear a gene that codes for a receptor for a host- specific toxin produced by a pathogen
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What EXACTLY causes cytoplasmic
resistance in non, TMS-corn?? |
non TMS corn lack the a mitochondrial attachment site for the T-toxin ( a host-specific toxin)
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"TMS" in TMS corn stands for....?
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Texas male sterile
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Overview of TMS corn epidemic?
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TMS cytoplasm (Texas male-sterile) is
inherited via mitochondria from the female plant and carried a susceptibility gene to the new strain “T” of pathogen, (Race O was the more common nonvirulent strain) -1970: 80% of all hybrid corn in U.S. contained TMS—led to epidemic |
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Define horizontal resistance.
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Characterized by many genes
controlling resistance, perhaps hundreds ! Resistance is expressed against ALL races of the pathogen uniformly, very little variation among races |
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Downside to horizontal resistance?
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Does NOT keep plants from becoming
infected, just slows down the development of the epiphytotic *economic threshold is not met. |
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there is always some level of ____ resistance inherent in a plant.
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horizontal aka "durable"
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horizontal resistance selects for traits like....
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- Small pustules
- Long latent period - Spores require a longer germination period, - Etc. |
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Define vertical resistance.
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-resistance controlled by one to a few major genes
• Gene product allows the plant to “recognize” a SPECIFIC race of pathogen |
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Vertical resistance is usually expressed in 1 of these two ways.....
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• Hypersensitivity (rusts, Soybean cyst/RK nema) -(extreme reaction or cell death at pathogen site)
• Immunity—essentially “non-host” resistance |
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What is gene pyramiding?
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a way of determining and introducing multiple genes which each impart resistance to an SINGLE pest, or impart resistance to a single pest through independent host pathways.
*Used in VERTICAL resistance. |
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benefits of vertical resistance?
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Genes can be effective for long periods with
monocyclic soil-born diseases: |
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downside to vertical resistance?
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Failure of vertical genes leads is commonly called the
“boom-and-bust cycle |
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Define the gene for gene hypothesis?
What phenomenon does gfg explain? |
• G-F-G simply states that for “for every gene in the
pathogen that confers virulence, there is a corresponding gene for resistance" • G-F-G helps explain the co-evolution of virulence and resistance |
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Who first proposed the gene for gene hypothesis?
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• Proposed and first proven by H. H. Flor from NDSU
working on Flax Rust, Melampsora lini • Basis of virtually all plant disease crop breeding programs. |
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what is the quadratic check?
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like a punnett square for pathogen virulence/avirulence, and a host susceptibility/ resistance. With heterogenous players, 3 of 4 will get the disease. The host must have the correct receptors for the pathogen to be resistant.
*A single mutation in the pathogen can allow the corresponding resistance gene to become ineffective, i.e., resistance has “broken down” |
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explain a/virulence and resistance dominance/ recessiveness in plant pathogens.
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Virulence is usually recessive, avirulence is dominant
! Resistance is usually dominant ! Disease can occur when plants LACK a resistance gene OR when the pathogen has the correct gene to overcome the resistance gene |
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Describe the cycle of resistance genes and virulence genes.
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pathogen carries effector E1
plant carries rare R1 resistnace R1 is amplified in population-selecting against E1. Lowered E1 results in selection against R1, then E1 can rebound. |
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If a pathogen produces an elicitor it is ______, if a pathogen does not produce an elicitor, it is ______.
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avirulent (plant can sense, then raise defenses)
virulent |
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What are quantitative trait loci?
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A region of DNA that is associated with or closely
linked to a particular phenotypic trait: often found on different chromosomes. QTL’s help us identify what specific genes code for a particular trait. |
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What is quantitative inheritance?
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(polygenic) trait requiring two or more genes.
eg. skin color |
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Knowledge of QTL's (quantitative trait loci) can help us determine how traits are structured in the genes on a spectrum between ______ and _______.
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1. Is the trait controlled by many genes of small
effect? Or, 2. Is the trait controlled by a few genes of large effect? |
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Marker assisted selection (MAS)?
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morphological, biochemical or nucleic acid variants are used to
indirectly select for a trait of interest. Only detects pathogen PRESENCE, not quantitative. NOT selecting for the trait itself, but a marker genetically linked to it |
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What are constitutive defenses (general)?
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Always “on” or present; passive…
eg, cuticle, sclerenchyma, secondary metabolites |
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What are Induced defenses?
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Active, respond to a specific attack…callousing, induced lignification, silicon deposition, etc.
eg. |
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easiest way to infect host with engineered genes?
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Agrobacterium - best to find a host that agro can infect- makes things easier!
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mobile "jumping genes" found only in primates are ...?
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ALU elements
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Problems with Ti (Tumor inducing) plasmids- How are they dealt with?:Actually an “oncogene” = cancer
causing gene |
Use a “disarmed” Ti vector, this means
that you simply remove the tumorinducing genes (& other cumbersome extra genes) |
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Problems with Ti (Tumor inducing) plasmids- How are they dealt with?:“Promoter” problems, i.e., it has a
“bacterial” rather than a “eukaryotic” promoter to turn the gene on |
Use a “35S Cauliflower Mosaic Virus”
promoter |
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Problems with Ti (Tumor inducing) plasmids- How are they dealt with?:Host range restricted primarily to dicots..4 strategies...
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1. Use a “gene gun”, biolistic DNA delivery
2. Use protoplasts -Electroporation (increases cell wall pereability) -Protoplast fusions with compatible species (essentially liposomes- cell wall removed) 3.Virus vectors -problems with host range and gene size 4 Modified Agro strains |
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What is triparental mating?
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a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second bacterial strain into a third bacterial strain
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-Electroporation
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increasing a cell wall's permeability
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-Protoplast
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cells with cell wall removed- essentially a liposome (contained by lipid bilayer)
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Genetic engineering limitations: You can only clone_______ resistance
genes (Identifiable ones) BUT, _____ may allow us to go after ___________ genes with more confidence. |
VERTICAL
QTL’s HORIZONTAL |
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What kinds of genes are we moving into plants?
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1. Resistance:
-viral coat resistance -ceropins toxins from amphibian skin 2. nutritional alteration 3.stress tolerance 4. fruit ripening (eg FlavR Saver) 5.antibodies..Edible vaccines! 6.therapeutic agents 7. polymers for biodegradable plastics 8.Lignin content alteration for easier plant processing (paper, fuel, etc) |
