Ames Test

what will you see in the positive control disc (contains mutagen)?

# of colonies around disc: higher rate of back mutations because they were induced by a chemical

what will you see in negative control disc (contains water)?

# of colonies on plate: rate of spontaneous back mutations

decribe the ames test procedure

- streak a lawn of his- salmonella on minimal media -add paper disc soaked w/ test chemical and let bacteria grow

if round-up didn't increase the # of colonies in our ames test, does that mean it doesn't cause cancer? what are some explanations?

-concentration not high enough -concentration too high (could've had a toxic effect on bacteria near disc... can't see back mutation if dead) -real ames test done including liver enzymes to stimulate what would happen to chemical inside the body -our strain M only tests if chemicals can cause substitution mutations (not for insertions/deletions)

describe round-up safety

-even if something is nontoxic, it could still potentially cause cancer over long periods of time -2015-2018: conflicting studies about whether glyphosate is associated with non-Hodgkin lymphoma in humans *who's cancer research arm (IARC) says probably causes cancer *EPA says unlikely to cause cancer -several lawsuits have been won against Monsanto (EX: farmers exposed to a lot of round-up)

describe salmonella strain M

-has a missense mutation in the his gene -for this we must use a mutagen (NaAz) that is know to cause base substitution mutations b/c it could revert the changed base in strain M back to the correct base

define back mutation

-reverts a mutated gene back to its original state -fixes the mutated his gene allowing it to produce histidine

describe the toxicity test

-we can test if our chemicals are toxic to bacteria -uses complete media (CM) plates that have histidine in them (kill)

what will happen if the test chemical on your plate is a mutagen?

-will make random mutations in salmonella's genome -possibility of a back mutation

how would we test if round-up is toxic to humans?

-you could grow human cells from a test tube or a culture disc and expose them to whatever chemicals you want and see if they die -you can also use animals that are similar to humans to do these toxicity tests

if round-up did yield a high # of colonies, what else would we need to show that round-up causes cancer in humans

-you could grow human cells from a test tube or a culture disc and expose them to whatever chemicals you want and see if they die -you can also use animals that are similar to humans to do these toxicity tests -you could also look at studies of humans: groups of humans that have been exposed to large amounts of round-up through their work vs groups of humans who have not been exposed to round-up and see who has more cases of cancer

How would we test if round-up causes cancer?

-you would need to expose that chemical over long periods of time to an animal that could get cancer such as mice -you could also look at studies of humans: groups of humans that have been exposed to large amounts of round-up through their work vs groups of humans who have not been exposed to round-up and see who has more cases of cancer

describe the example results from the toxicity test

1. Water Control (- control): no zone of inhibition 2. Sodium Azide (+ control): no zone of inhibition 3. test chemical (round-up): yes; zone of inhibition present @33 for a visual

describe the example results from the ames test

1. Water Control (-): had 24 colonies (around this means that it's not a mutagen) 2. sodium Azide (+): had 41 colonies (around this or higher means that it's a mutagen) 3. Test Chemical (Round-up): had 23 colonies (ask yourself during a test if that # was closer to the - or + control. In this case it's closer to - control so your conclusion would be "the test chemical doesn't cause substitution mutations go to @30 secs for visual

describe our test chemical: round-up (glyphosate)

1. inhibits an enzyme that plants use to synthesize certain amino acids -reduced protein production ---> plant death -bacteria have the same enzyme but mammals/humans do not (selective toxicity) 2. widely used herbicide to kill weeds/grass -crop plants can be genetically modified to be resistant (how?) *point mutations in genes encoding the affected enzyme ---> round-up can no longer bind to enzyme

describe his- mutant and MM plates which are used in the Ames test

1. uses "his- mutant" salmonella typhimurium bacteria -has a mutation that makes his enzyme nonfunctional *his enzyme normally makes histidine amino acid *his mutant salmonella can't make their own histidine 2. uses minimal media plates -do not contain histidine

A) explain how the ames test works B) what is a back mutation? C) how can BM's occur?

A) a filter paper is saturated w/ a sample chemical and placed on minimal media plate that is spread w/ his- mutant salmonella typhimurium bacteria -mutagenic = will induce back mutations in some his- bacteria causing them to become his+ and grow on MM plate -nonmutagenic = no growth or spontaneous back mutations B) revert the changed base in strain M back to the correct base C) by certain mutated genes

A) our salmonella strain M has what kind of mutation? -Normal: GTCGATC(T)CGGTATT -Mutation: GTCGATC(C)CGGTATT B) what would be the back mutation that would fix this his gene? C) what kind of mutation would this be? (base substitution, insertion, or deletion)

A) missense mutation B) C ---> T C) base substitution

explain what the CM plates tell you about each of the chemicals that we used for the toxicity test A) water B) NaAz C) test chemical

A) no zone of inhibition = not toxic to bacteria B) no zone of inhibition = not toxic to bacteria C) zone of inhibition = toxic to bacteria

explain what the MM plates tell you about each of the chemicals that we used for the ames test A) water B) NaAz C) test chemical

A) rate of spontaneous back mutations B) causes base substitutions and possibility of back mutations C) if a mutagen: will make random mutations in salmonella's genome and possibly a back muation if not a mutagen: spontaneous back mutation

what test would we use to find out if roundup causes mutations?

Ames test

explain how the CM plates and the MM plates differ from each other

CM plates have histidine, MM plates don't contain histidine

what would we see if a back mutation occurred?

bacteria regain ability to grow on minimal media (MM) plate because they can make their own histidine

which type of mutation was strain M used to detect?

base substitution mutations

which type of mutation does sodium azide induce?

base substitutions and possibly back mutations in strain M

how are the terms mutagen and carcinogen related?

because many mutagens are also carcinogens as the mutations they induce can cause cancer over time

who developed the ames test?

bruce ames

many mutagens are carcinogens. Define carcinogen

capable of causing cancer

define mutagen

chemical capable of altering the nucleotide sequence of DNA

what does "his- mutant" indicate

it's nonfunctional

what does growth on an MM plate indicate?

mutagenic substances caused some organisms to mutate and grow

did the test chemical cause mutations? explain how you know

no; b/c it had the same # of colonies as our negative control meaning that it only produced spontaneous back mutations

what does no growth on MM plate indicate?

nonmutagenic substance that doesn't cause organisms to mutate

is round-up toxic or nontoxic to humans?

nontoxic; humans don't have the enzyme that is targeted by round-up (selective toxicity)

what is our known mutagen and what does it do?

sodium azide: causes base substitutions (could possibly cause a back mutation in strain M)

explain the difference btwn a spontaneous and an induced mutation. Which of your test plates contains colonies produced by spontaneous mutation? which contains colonies produced by induced mutations?

spontaneous mutation: accidental mistakes when DNA replicates itself Induced mutation: exposure to chemicals such as roundup MM plate w/ NaAz produced colonies w/ induced mutations MM plate w/ water produced colonies w/ spontaneous mutation

what will happen if you place the salmonella typhimurium bacteria on a minimal media plate w/ no histidine?

the his- mutant shouldn't grow

what do colonies that form in the non mutagenic plate tell us?

the rate of spontaneous mutations in our system

what is the purpose of the Ames test?

to determine if a chemical is a mutagen

what test would we use to find out if roundup is toxic to bacteria?

toxicity test

if round-up caused a zone of inhibition in the toxicity test, was that expected

yes; it is known that bacteria do have the enzyme that round-up inhibits

if the test chemical is not a mutagen, could there still be colonies?

yes; this is because of spontaneous mutations (you can see this in our negative control which was water)

describe the expected results on complete media from the toxicity test

zone of inhibition indicates that the chemical was toxic to bacteria (this does not mean that it's toxic to us)