1. UNIQUE FEATURES OF BACTERIAL GENETICS
· Single genome per cell
· Fast growth rates --- > rapid results
· Enormous numbers of offspring; even the most improbable events can occur at significant
    rates

2. TERMINOLOGY
Mutation and mutation rate

• mutation = inheritable changes in the sequence of nucleic acids.
• mutant = an organism with these changes.
• mutagens = substances that induce mutation. Can come about as a result of single base
    change, multiple base changes, even addition or deletion of large amounts of DNA

3. WILD-TYPE AND MUTANT
A mutant will be different from its parent (wild type), its genotype or genetic make up has
    been altered. The phenotype or visible properties of the mutant may or may not be altered
    (what is observed: red pigmented colony, resistant to antibiotic, requirement of leucine for
    growth)

· Wild-type is capable of full range of metabolic activities found in type specimens. Ex: wt E.
    coli can manufacture all 20 amino acids from single C-source, manufacture all vitamins,
    fatty acids, vitamins, etc.
· Mutant could be defective in synthesis of some substance, e.g. amino acid leucine (leu^-
    strain); would have to be fed leucine in order to grow.

3. Nomenclature for Phenotype and genotype
· Genotype = 3 small italics letters code plus a capital letter indicating the gene involved in
    the process: lacZ (indicates the gene for Lactose Z protein)
· Phenotype = is indicated by a 3 letter code that ends in a +/- (Thr+ = strain that can make
    its own threonine)

· Don't name wt genes, only mutants: e.g. E. coli B leu^- thr^- lac^- penR (but not E. coli K12 leu+ thr+   lac+ penS)

4. MECHANISMS OF MUTATION
Two types: a) Spontaneous and b) Induced

a). SPONTANEOUS - Originate from lesions in the DNA as well as from replication errors.
· can arise from diverse sources; don't know exact cause of any specific mutation. Errors in
    base pairing occur, even after proofreading, with frequency of 1 in 10⁶ to 1 in 10⁷.

b). INDUCED:
A) base analogs
· compounds like 5-bromouracil looks like Thymine, get incorporated into DNA during
    replication.
· However, when serving as templates, they don't always form the "correct" match with A,
    instead sometimes pair with C.

B) Alkylating agents
· Changes base structure and alters base pairing.
· Nitrosoguanidine - adds methyl group to guanine, and instead of paring with Cytosine, mispairs
    with Thymine

C) Interkalating agents:
· Certain chemicals called intercalating agents can slip into DNA double helix between base
    pairs, induce mutations that result in extra bases being added. Resulting genetic code now
    has extra base, will be frame shifted at some point. Protein is made, but typically garbage,
    has no relation to original protein, often has "stop" codon earlier than wt gene, causes
    truncated garbage proteins.
· Acridine dyes (acrydine orange) and ethidium bromide are good intercalating agents, cause
    frameshift mutations.

D) UV radiation
     Causes fusion of adjacent thymine residues in same strand --- > thymine dimers. Can be
        repaired, but if not will cause inconsistent base insertions during replication.

Note 1: all of these approaches often produce multiple mutations in different genes

Note 2: these mutations can potentially be reversed by a second mutation which substitutes the
    original base for one altered to produce initial mutant. Such mutants are called revertants.
    Most mutations can revert with some frequency, even if slight.

D) INSERTION MUTAGENESIS
· Transposons are movable genetic elements, flanked by insertion sequences. When
    transposon moves, can insert itself within a structural gene. Like taking sequence
    XYYWWLALL and moving in into coherent phrase; FOURSCORE AND SEVEN ....
    Result: FOURSCCXYYWWLALLORE AND SEVEN ....

This is gibberish, destroys sense of a word. Similarly, inserted DNA will be transcribed, and disrupt the normal protein.

 · Value of transposon mutagenesis: can get a single insertion (rather than a cluster of mutants
    as in chemical mutagenesis); can actually find site of mutation on gels after digesting DNA
    with restriction enzymes.

4. POINT MUTATIONS: (usually involves a single base)
a) Silent mutation: CGU to CGC = Arginine (normal protein)
b) Missense mutation: single base substitution  GAG (Glu) to GUG (Val) (faulty protein)
c) Nonsense mutation: sense to stop codon (UAA, UAG, UGA) (incomplete protein)

5. FRAMESHIFT MUTATIONS: (usually involves more than 1 base)
Results as a consequence of insertion or deletion of bases (faulty protein)

6. TYPES OF MUTANTS AND SELECTION STRATEGIES
Colony morphology: Easy to detect (e.g. colony smooth rather than slimy); often reflect
    changes in genes affecting cell surface. This is too easy and in most cases there are not phenotypic
    changes!

1.) Resistant mutants
Easy to select; add an antibiotic or a virus, look for zones where most cells are inhibited, a
    few mutants can resist agent and will grow.

2.) Auxotrophs
Auxotroph = mutant that cannot grow on minimal medium, requires certain supplement(s).
    (Prototroph= wild type, it will grow in minimal medium or medium lacking the supplement)

Many auxotrophs have been isolated. Very useful in figuring out metabolic pathways

Example: leu- auxotroph, can't grow without added leucine. How to select such a mutant?

Screen for mutants: spread cells on a plate containing leucine so mutants and wt will both
    grow (can't yet tell which is which). Do replica plating and transfer to two plates: (1) No leucine,
    (2) Yes leucine.  Now incubate. If colony grows on plate (2) but not (1), it is a desired mutant.
    Can't pick from (1) (it's not there, remember?) but can pick from comparable site on plate 2.
    Store this colony, give it a mutant number, repeat as long as needed to get reasonable
    number of mutants.

Problem: mutant frequency is low. Might have to screen 10,000 plates just to find a single mutant.
    Too much work!! Need better odds........USE MUTAGENS!!!

3.) SUGAR FERMENTATION MUTANTS
You can use MacConkey agar to identify lac- mutants (cannot use sugar lactose for growth).
    MacConkey agar contains other nutrients, so all cells can grow; but contains significant
    amount of lactose and pH indicator. If cell can use lactose, will produce acid (fermentation
    will occur even on colony when oxygen is exhausted during vigorous growth) and colony
    will turn red. If colony remains white, it is a lac mutant.

Note 1: designation lac- looks similar to leu-, but misleading.
· lac- means "can't use lactose" -- this is not an auxotroph.
· leu- means "can't manufacture leucine" -- this is an auxotroph.

Note 2: Mutants can be Lac- for a couple of reasons. (1) can't make enzyme beta-galactosidase
    needed to break down disaccharide. (2) can't make permease needed to get substrate across
    membrane, but can still degrade sugar.

4.) CONDITIONAL LETHAL MUTANTS
    For any gene, possible to get mutations that affects protein folding. Some of these will cause
    protein to denature at modestly high temperatures (e.g. 42^oC), whereas protein will be
    stable at cooler temps (30^oC). These are called temperature sensitive mutants, one example
    of a conditional lethal mutant (lethal under one set of conditions, not under another)

If such mutations occur in gene absolutely required for cell survival, then at higher (restrictive) temperature, protein will unfold and cell will die. At lower (permissive) temperature, protein folds normally and cell can grow.

Easy to select: (1) mutagenize; (2) grow cells at 30^oC, plate out colonies. (3) use replica plating into two plates. (4) growth at 30^o and at 42^o. Pick colonies that survive 30^o but die at 42^o.

Result: can isolate a large class of mutants that are temperature-sensitive. These mutants are entirely distinct, except that all affect proteins critical for survival.

Study individual Ts mutants. Can discover many genes and their protein products involved in critical cell processes such as cell division, DNA replication and separation, RNA synthesis, etc.

5. AMES TEST
An application of power of bacterial genetics to help screen for substances that might cause cancer.

CARCINOGENS
Many substances can cause cancer: large number of chemicals, radiation, etc. Wide variety, but common denominator in general is that almost all carcinogens cause mutations in DNA. When critical cell targets regulating cell division are mutated, result is cancer. Can occur in any tissue.

DIFFICULTIES WITH CARCINOGEN TESTING
    NIH has protocols for testing suspected carcinogens. Requires special strains of inbred animals (genetically homogenous), different dose levels, multiple repeats, statistical analysis, various techniques for assessing presence or absence of tumors in each animal.

· Very expensive: can cost anywhere from hundreds of thousands to millions of $$, take from 6
    months to 2 years to test
· Thousands of new chemicals are introduced to U.S. industry each year, find their way into
    cosmetics, foods, drugs, consumer products. Impossible to screen most of these for possible
    carcinogen activity.
· But could screen for mutagenic activity; take chemicals that show up positive, screen those
    for carcinogenic potential. A very efficient strategy; bacteria are cheap, quick.

DESIGN OF THE AMES TEST
· Bruce Ames developed test using histidine auxotrophic mutants of Salmonella typhimurium
    (cousin of E. coli. You can also use a E. coli Trp auxotroph)
· Assumption: carcinogens are also mutagens ----- in most cases this is correct!!
· Ames tester strains are his- point mutants (possibility of reversion mutation is there).
· Reversion mutation (revertant)

Test design:
(1) Control Plate: spread ~10⁷ his auxotrophs on a plate containing minimal medium, lacking histidine
    (his-).

Result: cells won't grow, except for occasional revertant spontaneous mutant (at 1 in 10⁶ rate, expect ~ 10 mutants/plate).

(2) Experimental plate: spread same cells on similar plate, add a filter disk soaked in test chemical
    solution. If chemical is mutagenic, will diffuse into agar, will see increased number of mutants
    surrounding the disk.

Note: actually this test as just described will miss many chemicals that are mutagens in animals. Why? In animals, chemicals are detoxified in liver, often by many chemical steps. In process, some chemicals which are not initially mutagenic are converted into mutagens. To expand scope of Ames test, must add preparation of liver enzymes (made by grinding up fresh animal liver, centrifuging out debris) = liver microsomal fraction. With this addition, many more chemicals show up as Ames positive.

Economics of Ames test: costs only a few $ 100, instead of millions. Takes only a couple of days, instead of a year or more. -90% of chemicals that test positive for mutagenesis have been found to be carcinogenic in animals. There are some carcinogens that don't show up as mutagens on Ames test, so it's not foolproof, but a good screen.

Many industries now routinely use Ames test as screening for new products, will not develop products further if positive test (good practice in the age of soaring liability costs).