LECTURE 4 (Pathways) Flashcards
What are prototrophs?
organisms able to grow and reproduce in mm and have all the metabolic pathways to reproduce
What are auxotrophs?
individuals that have some requirement for reproduction e.g some aa
How can we isolate auxotrophs?
- filtration enrichment you take cells and breed them in mm with a mutagen. the ones that mutagenizes will grow whereas the others won’t. culture over night and then with a pore membrane will select the ones that are small and haven’t grown which are the auxotrophs
- killing enrichment, you use antibiotics that is called nystalin and kills growing ones faster than non-growing ones. then remove the dead ones and plate the non-growing ones in a solid medium then put them (colonies) in individual tubes) and test them for different auxotrophic conditions
How can we know which is the requirement of an auxotroph?
you compare with databases its DNA sequence
What are alleles?
alternative forms of some gene
you have two loci involved in histidine synthesis his a and hub b with 3 alleles at each locus. (hisa+, hisa-1 and hisa-2)
same for b. are these allelic?
no because they’re located in different loci
what is the best test for testing allelism?
cis/trans test
What is the cis/trans test?
most specific test for allelism it tests whether two alleles are in the same genetic unit of info (the cistron)
How can we obtain specific types of auxotrophs such as Arg-?
to get them you need mutations in the Arg- gene. you add all necessary nutrients (bur arginine)to a plate and all mutants will grow but the arg-. then do a filtration/killing enrichment technique to select the auxotrophs that will be this time arg-
How can we use mutants to determine biochemical pathways?
you have this pathway A—-B—-C—D D being tryptophan. if any of the enzymes catabolizing the steps is not functional then tryptophan won’t be produced by the biochemical machinery of the organism and therefore it’d be a tryp mutant. if enzyme 2 is not working then AB can be produced and CD but not BC we can plate the mutants in different plates and provide B an C and if they grow that means that’s the specific enzyme that’s missing because we plate them on mm.
What is negative feedback on a pathway?
It’s when the end product has been produced in too much quantity so it ‘tells’ the 1st enzyme to stop synthesizing.
How can we know exactly which enzyme is defective?
because normally if an enzyme is defective the product before it gets accumulated and usually secreted into the medium so we can know which enzyme was however we need to know the biochemical pathway to know which are the products
Whats a cross-feeding test for?
it’s a test for seeing how a pathway is organized
give an example of mutants with double requirement.
mutants that require met and tyr
How can we make mutants with double requirement grow?
we plate them with both aa or in the case of met and tyr if we plate them with homoserine they’ll grow as well.
