- Single genome per cell
- Fast growth rates ---> rapid results
- Enormous numbers of offspring; even the most improbable
events can occur at significant rates
- Genome can be sequenced in reasonable time
- mutation = heritable change in DNA (or RNA for
- can come about as a result of single base change,
multiple base changes, even addition or deletion of large amounts of DNA
- effects of a mutation range from being
- undetectable (e.g., base change leads to a same amino
acid being specified, or slightly different amino acid with no effect on
protein structure or activity)
- slight (e.g., amino acid substitution leads to altered
protein, slight change in active site)
- severe (e.g., amino acid substitution alters active
site, enzyme no longer binds substrate)
- lethal (e.g. mutation affects a critical enzyme such
as RNA polymerase)
- 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,
- 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.
- Phenotype = what is observed. Ex: red pigmented
colony, resistant to antibiotic, requirement of leucine for growth
- Genotype = symbolic representation of gene
responsible for phenotype. Ex: leu-, strR
- Typically use 3-letter codes for genotype.
- Every isolate should be identified by strain
designation; e.g. E. coli K12, E. coli B
- 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)
- Collections of strains are kept.
- Major U.S. source is ATTC (American
Type Culture Collection); can order any strain for minimal fee.
- ATTC maintains the following frozen or freeze-dried
stocks: (number of species in parentheses)
- Algae (120)
- Bacteria (14400)
- Fungi (20200)
- Yeasts (4300)
- Protozoa (1090)
- Cell lines: animal
- Cell lines: plant (25)
- Viruses: animal (1350)
- Viruses: plant (590)
- Viruses: bacteria (400)
- Yale Univ. keeps the definitive E. coli
collection (in freeze-dried form in envelopes); can search computer database,
identify specific strains to order.
- "spontaneous error": 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 107
to 1 in 108.
- base analogs: compounds like 5-bromouracil look
like Thymine, get incorporated into DNA during replication. However, when
serving as templates, they don't always form the "correct" match of A, instead
sometimes pair with C.
- nitrous acid: causes deamination; resulting bases
can cause incorrect base pair matches
- 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.
- 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 are good intercalating agents,
cause frameshift mutations.
- Note: to get frameshift revertants, typically have to
use a frameshift mutagen.
- Can result from ionizing radiation, other treatments
that cause double-stranded breaks. During DNA repair, section of DNA can fail
to get reintegrated ----> deletion of dozens, maybe thousands of base pairs of
- Deletion mutants cannot revert. Very stable mutants.
- Transposons are movable genetic elements, flanked by
- When transposon moves, can insert itself within a
- Example: Like taking sequence
XYYWWLALL and moving in into coherent
phrase; FOURSCORE AND SEVEN
.... Result: FOURSCXYYWWLALLORE
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
- Easy to detect (e.g. colony smooth rather than slimy);
often reflect changes in genes affecting cell surface
- 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
- Auxotroph = mutant
that cannot grow on minimal medium, requires certain supplement(s).
- Many auxotrophs have been isolated. Very useful in
figuring out metabolic pathways
- Example: ser- auxotroph, can't grow
without added serine. How to select such a mutant?
Naive selection procedure for auxotrophs
- Screen for mutants: spread cells on a plate containing
serine so mutants and wt will both grow (can't yet tell which is which).
- Locate isolated colonies. Pick each colony, transfer to
- no added serine
- + added serine
- Locate colonies so can compare same colony on each
- 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.
- Enrichment culture. Need to find a way (enrichment
culture) to enrich for serine auxotrophs in original culture. But how? serine
auxotrophs can do everything wild type can do except grow in serine.
- Solution (devised by Joshua Lederberg): penicillin
selection. Penalize cells that can grow, reward cells that can't grow.
Penicillin selection procedure for auxotrophs
- mutagenize culture to increase mutation frequency
- grow cells in minimal medium supplemented with 19 amino
acids (but not serine), vitamins, other biosynthetic needs for which
auxotrophs other than serine have needs. Result: all cells, both wt and other
auxotrophs, will grow. But serine auxotrophs can't grow.
- when cells are happily in exponential growth, throw in
penicillin (or other cell wall antibiotic). Will lead to wall damage in all
growing cells, but not in serine auxotrophs.
- after lysing cells, plate survivors on plates containing
serine, use two plate screening technique described above. Should enrich for
serine auxotrophs considerably.
- Note: same type of procedure can be used to select any
auxotroph. In practice, try to find a lot of independently isolated mutants,
then study and compare them (some will be in same gene).
Conditional lethal mutants
- For any gene, possible to get mutations that affect
protein folding. Some of these will cause protein to denature at modestly high
temperatures (e.g. 42o C), whereas protein will be stable at cooler
temps (30o C). 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:
- grow cells at 30o, plate out colonies.
- test each colony for growth at 30o and at
- Pick colonies that survive 30o but die at
- 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.
An application of power of bacterial genetics to help
screen for substances that might cause cancer.
- 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
- 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.
- Bruce Ames developed test using histidine auxotrophic
mutants of Salmonella (cousin of E. coli)
- Ames tester strains are his- point mutants
(possibility of reversion mutation is there).
- Test design:
- Control Plate: spread ~ 108 his auxotrophs
on a plate containing minimal medium, lacking histidine. Result: cells won't
grow, except for occasional revertant spontaneous mutant (at 1 in 107
rate, expect ~ 10 mutants/plate).
- 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).