Microbiology 101 – Flashcards
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Point Mutation |
A mutation where a single base is changed to a different base. |
3 types of point mutations |
1. Missense muation 2. Nonsense mutation 3. Frame-shift mutation
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Missense mutation |
Change produces a different codon on the mRNA, leading to a possible change in amino acid sequence of protein |
Nonsense mutation |
The change produces a stop codon |
Stop codons |
UAA, UGA, and UAG (no tRNAs) |
Frame-shift mutation |
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; ; Reading frame |
; ; The codon triplets |
; ; 3' end of mRNA |
Encodes C-terminal region of proteins and can be mutated without loss of function. |
Deletion |
Loss of DNA segment |
Insertion |
Insertion of a few bases in DNA replication |
Insertion in bacteria |
Made by viruses or moveable segments called transponsons or insertion sequences. |
X-Rays |
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Inversions |
Sequence flipped backwards |
UV Light |
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Base analogs |
Chemicals whose structure is so similar to specific nucleotides that DNA polymerase accidentally inserts them into DNA when replicating
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Hydroxylamine |
Chemical that changes cytosine structure
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Nitrous Acid |
Chemical that changes cytosine and adenine structures |
NTG |
Chemical that produces alkylation of bases at the replication fork (grilling meat) |
EMS |
Chemical alkylating agent |
Acridines |
Organic dyes that have a flat planar structure that slides in between the base pairs in DNA causing errors in replication / repair. |
Adenine pairs with thymine (mutation) |
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Screening |
Observe and dest colony to identify mutatants that have...
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Phenotype |
Describes appearance of organism |
Genotype |
Actual genetic make-up of organism |
Direct selection |
Set up a condition in which only mutants can grow |
Indirect selection |
2-Step process by which first all cells except mutant are killed and secondly the desired mutant grows |
Penicillin Selection - Step 1 (indirect selection method) |
Treat 109 bacteria with a mutagen like NG or UV light |
Penicillin selection - Step 1 effects |
; |
; ; Auxotroph |
; ; Nutritional defect that causes inability to prodcue histidine |
; ; Penicillin selection - Step 2 |
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Mutation probability during replication (per base pair) |
10-9 |
Is replication efficient? |
Yes, few errors. |
Probability of mutation creating new gene function |
10-10 to 10-12 |
Mutagen |
Agent that causes mutation and increases mutation rate 1,000 fold |
Mutagen / Carcinogen (relation) |
85% of carcinogens are mutagens and 90% of mutagens are carinogens |
Teratogen |
Chemicals that cause embryonic defects during development |
Ames Info |
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Ames Test
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Ames Results |
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; ; Spontaneous rate of reversion |
; ; Rate at which mutations spontaneously mutate back to normal |
; ; Coffee mutations |
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; ; Plasmid |
Circular, double stranded DNA containing 5 to 75 genes completely separate from chromosome that replicate on their own schedule and separate in DNA |
; ; 5 Classes of plasmids |
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; ; Conjugative plasmid |
; F-factor is contained on plasmid and containes genes that build the conjugation tube between donor / recipient cells in a mating-like event |
; ; R Plasmids |
; ; Plasmids carry genes that inactivate antibotics |
; ; Colicin plasmids |
; ; Plasmids that kill other bacteria |
; ; Virulence plasmids |
; Plasmids that produce a toxin that damages the tissues of the host or another protein essential for pathogens. |
; ; Degradative plasmids |
; Plasmids that encode enzymes that permit the bacterial cell to use unusual chemicals as a carbon source |
; ; Bioremediation |
A bacterium containing a degradative plasmid that can be added to contaminated soil (along with N and P) for nutrients that breaks down pollutants. |
; ; Conjugation |
; ; Method of transfer of one bacteria's plasmid to another |
F- strain |
Recipient strain that receives plasmid |
Conjugation bridge / tube |
Structure formed by extension of pillus of F+ to F- cell that is controlled by genes contained in the former |
After conjugation |
Donor still has copy of F+, but recipient also gets a cop and is now F+ as well |
Rolling circle (1) |
Type of DNA replication where a nick is formed in one of the two F-factor strands which open allowing DNA polymerase to bind and extend the nicked strand by using the unopened strand as a template |
Rolling circle (2) |
New strand replaces nicked strand which rolls of plasmid and the single stranded F passes through conjugation and into the F- cell |
Rolling circle (3) |
F- cell synthesizes complimentary strand and ends join to form two F+ cells. (Only F-factor is transferred, no bacterial genes on chromosome) |
Hfr strain |
When the F-factor inserts itself into the bacterial chromosome it creates this cell that can transfer the entire bacterial chromosome into recipient cells. |
Hfr and F- recombination mating |
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Quiet necessary for entire transfer (Hfr) |
100 minutes |
How did Hfr and F- mating lead to genetic mapping? |
The varying amounts of gene transfer (because when interrupted, only part transfers) correlated with conjugation time led to the first genetic mapping. |
Recombiant DNA methods |
Use of plasmids to isolate genes |
Cloning methods |
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Restriction endonuclease (definition) |
Enzymes that recognize and bind to specific sequences on DNA molecules |
Restriction endonuclease (method) |
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; ; Vector (definition) |
; ; For cloning, the vehicle for moving DNA from one source into a cell |
; ; Vector (method) |
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Transformation (definition) |
The act of getting the host bacterium to pick up the vector |
Transformation (method) |
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; ; Colony hybridization (method) NOT possible for human genes |
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; |
; ; DNA probe |
; Specific and complementary to the sequence you're looking for that contains a radioactive 32P marker |
Types of expression interest to yield a protein depended screening |
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Complementation |
Wild type gene inserted into a mutant strain to express a function; doesn't work if you want to express eukaryotic gene in a bacterium because eukaryotes have introns that are irremovable by bacteria |
; ; Enzyme activity / bioassay (method) |
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; ; Enzyme activity / bioassay (example) |
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Radioactive antibody assay (definition) |
Used to find bacteria that are producing a protein from a cell via purifying protein from natural source |
Radioactive antibody assay (method) |
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Cloning and expression in eukaryotes (problems) |
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Cloning and expression in eukaryotes (remedies) |
Remove introns by isolating mRNA and converting into cDNAs and then screening by some method to detect protein producing bacteria |
How to isolate mRNA |
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; ; cDNA |
; ; "Copy" DNA |
; ; Expression vector |
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; ; Immune system |
; ; Protects host against invading organisms |
; ; Two immune system parts |
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; ; Antibody (Ab) |
; Molecules made by immune system designed to recognize antigens presented by a foreign particle |
; ; Antigen |
; Protein, nucleic acid, carb, lipid, or any molecule present that elicits and immune response. |
; ; Epitope |
; ; Region of antigen that binds to antibody; 4-6 amino acids for proteins |
; ; Opsonization |
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; ; Leukocytes |
; ; WBCs that originate from primitive "stem cells" and can be differentiated |
; ; Two types of Leukocytes |
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; ; Phagocytosis |
; Engulfing of foreign particle by macrophage, neutrophiles, or other phagocytic cells of the immune system (endocytosis) |
; ; Phagosome |
The compartment of the cell containing the engulfed bacterium where the oxidative burst takes place via the oxygen radicals made by superoxide dismutase |
; ; Complement |
Group of proteins that bind to opsonized bacteria and through a series of reactions kill the bacteria by forming a pore in the membrane of the cell |
; ; Lymphoid stem cells produce... |
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; ; Myeloid stem cells produce... |
Myeloblasts which differentiate to form...
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; ; Neutrophil |
; ; Phagocyte found in blood that makes up 40% of myeloblasts |
; ; Basophil |
; Responsible for relasing histamine which induces an allergic response that makes up .1% of myeloblasts |
; ; Eosinophils |
; ; Participate in inflammation making up 1-7% of myeloblasts |
; ; Monocytes |
Responsible for stimulating immune system, including...
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; ; 5 classes of antibodies (produced by plasma B cells) |
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; ; IgG (info) |
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; ; IgG (picture) |
[image] |
; ; IgA |
; ; Similar to IgG except that they are dimers |
; ; Secretory Abs |
; Secreted by B cells of the mucous membranes of the body, eyes, UGI tract, lungs, and GI tract |
; ; IgM |
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; ; Resulting pore |
; 20 angstrom diameter allows contents to leak out via one IgM (efficient) or two close IgGs (not so efficient) |
; ; IgD |
; Not very well understood, produced by B cells and may be found on surface of B cells |
; ; IgE |
Produced by B cells that are reacting to allergies and binds to basophiles causing them to release immunostimulants, thus causing allergies and ridding body of protozoa and worms. |
Exons matched with antibody classes |
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; |
; ; 4 primary roles of antibodies |
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; |
; ; Abs construction |
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Heavy chain construction (1) |
Composed of V,D, and J exons and the constant regions exons (antibody exons)
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V exon |
Numerous variable exons; separated by introns in mice |
D exons |
Several diversity exons separated by introns |
J Exons |
Several joining exons of the mouse separated by introns |
Amino acid sequence of the constant region |
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Heavy chain construction (2) |
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Heavy chain construction (3) |
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; ; Light chain construction |
; Same genomic rearrangement determines light chain, and constant region is either K or upside-down y |
; ; Class switching |
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; ; How does body produce different Abs for each infection? |
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; ; Why do Abs have different functions |
They're specific for the antigen because they have the same variable region derived from same VDJ and and different functions based upon class which was determined by exon that encoded the constant region |
Clonal selection hypothesis |
Explaination for Abs specific for each antigen rather than all possible Abs when exposed to particular Ag because you don't want to make Abs that bonds to other viruses |
; ; How does colonal selection work? |
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; ; How do B cells differentiate and produce antibodies? (1) |
; ; |
; ; How do B cells differentiate and produce antibodies? (2) |
; |
Lymphokines |
; Proteins that stimulate the immune system produced by macrophage dendritic cells and T-4 helper cells and some activate B cells that are binding to ag moleucles while others activate cytotoxic T-cells |
; Macrophage and dendritic cells displaying pieces of a viral, bacterial, or fungal surface bind only to... |
... a subset of cytotoxic T-cells that have a receptor on their surface that recognizes the pieces of Ag displaced on the surface of the macrophage / dendritic cell |
; Cytotoxic T-cells that receive both signals from macrophage and T-4 helper cells... |
; Become activated and migrate throughout body and bind to cells of your body that are displaying the viral / bacterial Ag |
; ; Cytotoxic T-cells |
Bind to infected cells and kill them; 10s of thousands of different types, but only a small fraction activated during a an infection because most didn't bind to macrophage (even though they got "help") |
Bodily responses... |
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What causes autoimmune diseases? |
When the immune system inadvertently attacks antigens found on the surface of normal cells in our tissues |
Four types of hypersensitives |
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Practical uses of antibodies in the lab |
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Fluorescent antibodies |
Antibodies tagged with a small molecule that emits light when illuminated with UV or another wavelength of light that are used to detect specific bacteria or other cell types |
Examples of fluorescent antibodies |
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; ; FITC |
; ; Organic molecule that fluoresces when illuminated by UV light |
; ; Radioammune assay |
; ; Can measure amount of a molecule in a biological sample |
; ; ELISA |
; ; Enzyme linked immunosorbent assay used to detect antigen in samples |
; ; Western blot |
; ; Detects presence of a protein and used for diagnosis of viral bacterial pathogens |
; ; Hybridoma |
; Kohler and Milstein recieved nobel prize for this method of creating a cell that produces a single antibody |