Point Mutation

3 types of point mutations

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

• A base is inserted or deleted and the reading frame shifts, specifying wrong amino acids for proteins.
• Can add/delete 1 or 2 bases
• Can also changes stop codon.
• Can be disastrous outside of 3' end of mRNA

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

• Radiation breaks down DNA molecules
• DNA has repair mechanism that is error-prone and inversions can occur

Inversions

Sequence flipped backwards

UV Light

• Causes two thymidine bases next to each other in sequence to bond and form thymine dimer
• Repair process also error-prone

Base analogs

Chemicals whose structure is so similar to specific nucleotides that DNA polymerase accidentally inserts them into DNA when replicating

Hydroxylamine

Chemical that changes cytosine structure

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)

• Nitrous oxide removes amino group from adenine
• Turning into hypoaxanthine
• Similar to guanine
• DNA polymerase mixes the two up
• Matches it with cytosine
• Next replication continues, mutation is complete

Screening

Observe and dest colony to identify mutatants that have...

• Visible defects apparent by close observation
• Nutritional defects apparent by growing cells on different media
• Temperature defects apparent by growth in different temperatures

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)

Penicillin selection - Step 1 effects

• Results in 10⁴ his ^- for cells which won't grow without histidine in medium
• 10⁹ his⁺ cells w/ no mutation that will grow quickly in medium which thins peptidoglycan layer
• Cells are auxotroph
• - cells have original thick, while + are thin and swell and lyse

Auxotroph

Nutritional defect that causes inability to prodcue histidine

Penicillin selection - Step 2

• Recover the cells that didn't lyse and put them in fresh medium without penicillin
• Histidine can be added so the his^- cells grow on their own

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

• Histidine operon in salmonella 
• Double mutant histidine (--)
• Unable to synthesize histidine
• Spontaneous risk of reversion was 10^-12
• If mutagen is present, 10⁴
• Used reversion analysis to test chemical mutagens

Ames Test

• Plated 10⁸ double mutants on medium
• Slow growth
• DNA replication occurs
• Chemical (if mutagen) causes mutation that is double mutants, or revertants to his⁺, that grow on histidine deficient plates

Ames Results

• Chemicals known to cause mutations in rats and humans weren't mutagenic here
• Liver of humans can convert non-toxic into toxic
• By adding dog liver microsomes, this was remedied

Spontaneous rate of reversion

Rate at which mutations spontaneously mutate back to normal

Coffee mutations

• 1000 chemicals
• 17 of 22 tested cause mutations
• Single cup contains 10 mg of mutagens

Plasmid

5 Classes of plasmids

1. Conjugative
2. R
3. Colicin
4. Virulence
5. Degradative

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

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)

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

• Extends conjugation tube
• Entire chromosome begins rolling circle
• Half F-factor genes transferred first, all may transfer (depending on time)

Quiet necessary for entire transfer (Hfr)

100 minutes

How did Hfr and F^- mating lead to genetic mapping?

Recombiant DNA methods

Use of plasmids to isolate genes

Cloning methods

1. Restriction endonuclease
2. Vector
3. Transformation
4. Colony hybridization 

Restriction endonuclease (definition)

Enzymes that recognize and bind to specific sequences on DNA molecules

Restriction endonuclease (method)

• Restriction endonuclease Bam HI recognizes specific GATATC sequence and cuts ager the first G on the 5' end
• Frequency once every 4096 nucleotides Bam HI cuts once
• Human genome is 500 million base pairs, therefore about 120,000 Bam HI sites exist

Vector (definition)

For cloning, the vehicle for moving DNA from one source into a cell

Vector (method)

• Vector carries amp resistance and picks up foreign DNA
• Sticky ends connect
• Ligate to one another and no foreign DNA is picked up
• Donor DNA is sealed shut and won't grow b/c there is no replication

Transformation (definition)

The act of getting the host bacterium to pick up the vector

Transformation (method)

• E. Coli used as a host
• Via electroporation and CaCl₂ glycerol heat shock
• Once transformed, bacterium with foreign DNA isolated by plating with tetracycline (foreign doesn't grow)

Colony hybridization (method)

NOT possible for human genes

DNA probe

Specific and complementary to the sequence you're looking for that contains a radioactive ³²P marker

1. Complementation
2. Enzyme activity / bioassay
3. Radioactive antibody assay

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)

• Extract protiens from cell and ask if the enzyme is being expressed.
• Bioassay is used for harder cases w/ no protein in enzyme

Enzyme activity / bioassay (example)

• Neisseria gonnorheae that encodes protein allowing it to bind to UGI tract
• Transform gene into E. Coli, grow on epithelial cells
• If it sticks to epithelial cells like N. gonnorheae, it's expressed

Radioactive antibody assay (definition)

Radioactive antibody assay (method)

1. Plate transformed E. Coli colonies
2. Transfer to filter paper and lyse to release proteins\
3. Probe filter paper with 125 I-labeled Ab
4. The antibody sticks only to the debris of a colony that is producing the AB specific protein
5. Test for radioactivity similar to the colony hybridization method

Cloning and expression in eukaryotes (problems)

• Introns and exons transcribed into mRNA as a single protein
• Not functional because amino acids translated will act like giant insertions causing folind / no activity
• Stop codons also appear

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

• Use enzyme reverse transcriptase to copy DNA to a short primer that binds the poly A tail
• Add NaOH to break down mRNA
• Add DNA polymerase to extend hair-pin to create a complement
• Add S1 nuclease to clip off hairpin
• New double stranded DNA called cDNA with no introns and no promotors - can't be transcribed

cDNA

"Copy" DNA

Expression vector

• Allows genes to be expressed
• Has LAC promoter and LAC operator for regulating transcription
• Has terminator at transcription end
• Have ribosome binding sites so that message can be translated
• Also has cloning site that allows vector to open and cDNA to be put between promotor-ribosome region and transcription terminator

Immune system

Protects host against invading organisms

Two immune system parts

1. Humoral ammunity (Abs)
2. Cellular ammunity

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

• Comes from greek for preparing a meal
• Binding of bacteria by antibodies to prepare for phagocytosis or complement fixation

Leukocytes

WBCs that originate from primitive "stem cells" and can be differentiated

Two types of Leukocytes

• Lymphoid stem cells
• Myeloid stem cells

Phagocytosis

Engulfing of foreign particle by macrophage, neutrophiles, or other phagocytic cells of the immune system (endocytosis)

Phagosome

Complement

Lymphoid stem cells produce...

1. Pro-T lymphocytes (make t-lymphs)
2. Pro-B lymphocytes (make b-lymphs)
3. Plasma cells (make Abs)
4. Memory cells

Myeloid stem cells produce...

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...

1. Macrophages = phagocytic cells that wander in tissues
2. Histiocytes = fixed monocytes

5 classes of antibodies

(produced by plasma B cells)

1. Immunoglobulin G (IgG)
2. IgA
3. IgM
4. IgD
5. IgE

IgG (info)

• Half-life is 23 days
• Most abundant in AB blood
• Plays an important role in antitoxins, complement fixation, and transfer of immunity for mother to fetus

IgG (picture)

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

• 5 subunits with IgG structure
• First class made by immune response to new antigen
• Important for agglutination and opsonization
• Cause cell death via complement cascade

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.

• Alpha = IgA
• Gamma = IgG
• Delta = IgD
• Epsilon = IgE
• Mu = IgM

4 primary roles of antibodies

1. Opsonization of bacteria for phagocytosis
2. Virus neutralization
3. Toxin neutralization
4. Complement fixation

Abs construction

1. Amount of AB's in blood at different times of development
2. By age 2-3 months, a person will have all B cell types as they will ever have
3. Age 2-3 years a sharp increase in IgG and IgA is seen

Heavy chain construction (1)

Composed of V,D, and J exons and the constant regions exons (antibody exons)

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

• Determines the class of Ab
• C_u = IgMC_delta = IgDC_y = IgGC_epsilon = IgEC_alpha = IgA

Heavy chain construction (2)

• Recombination within gene occurs to join one D and one J
• Second recomb joins one V to the DJ complex
• Transcription occurs - L is the leader exon (L,V,D,J,C_u,C_y)

Heavy chain construction (3)

• Nuclear DNA becomes polydenylated at 3' terminus
• Splicing occurs to remove introns
• Translation produces heavy chain determined by VDJ section while Ab class is determined by C region

Light chain construction

Same genomic rearrangement determines light chain, and constant region is either K or upside-down y

Class switching

• Determines which Ab class is made
• In the rearranged genes, more recomb events occur as B gets older
• Instead of IgM, a different class of Ab is made depending on location of recomb in heavy chain

How does body produce different Abs for each infection?

• First Ab made is IgM that peaks after a few days and then declines
• IgG appears a few days later, produced by B cells as a result of recomb in the heavy chain gene, and stays for 21 days before declines
• Booster shots make both appear with little delay b/c of memory cells in the first immunization responding quickly to the second exposure

Why do Abs have different functions

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?

1. Macrophages and dendritic cells present antigens on their surfaces for the T-4 helper cells
2. T-4 helper cells bind to antigens displayed on the surface of macrophages
3. Killer T-8 cells kill other cells that display these ags

How do B cells differentiate and produce antibodies? (1)

• B cells form from stem cells in bone marrow
• Pro-B become pre-B
• Joining of the V,D, and J exons of the heavy chains occurs
• Immature B cells become mature B cells 

How do B cells differentiate and produce antibodies? (2)

• Mature B cells display IgM or IgD on surface
• If antigen binds to the IgM on the surface of this cell and recieves help from lymphokines secreted by macrophage and T-4 helper cells - it becomes B cells
• Multiplies rapidly; differentiate to become plasma cells which secrete large amounts of Ab into blood or memory cells.
• Some switch from IgM to IgG, and then from IgE to IgA. 

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...

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

Bodily responses...

• Depend on properties of pathogens
• Abs and cytotoxic T-cells are important for clearing viral diseases in the body
• Abs control bacterial diseases that do have an extracellular phase (cytotoxic T-cells are more important here)

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

1. Allergy
2. Cytotoxic
3. Immune complex
4. Delayed

Practical uses of antibodies in the lab

1. Fluorescent antibodies
2. Radioimmune assay
3. ELISA
4. Western blot

Fluorescent antibodies

Examples of fluorescent antibodies

• Bacterium X injected into animal
• Animal makes AB to bacterium X
• Isolate the IgG ab that is specific for bacterium
• Bind FITC to IgG
• Spread sample onto slide and dry, run a FITC labeled solution and UV to see if it's green (indicates host is infected)

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