Functional Complementation

Question 1

How can we isolate an arginine auxotroph? (Simple)

Case for Neospora?

Answer 1

Auxotrophs are unable to grow on minimal medium.

But can be isolated on minimal medium supplemented with arginine.

Using Replica plating.

= Likely unable to synthesise arginine as enzyme in its biosynthesis pathway has lost its activity from mutation in its corresponding gene

Neospora cannot use Replica Plating - mycelium
= isolate conidia in individual flasks.

Question 2

What is ESSENTIAL for a complementation assay, that should be carried out before?

Answer 2

The mutant phenotype HAS to be recessive.
Functional Complementation assay will not work if the mutant phenotype is dominant.
(In haploids, it will be dominant ofcourse, but needs to be recessive in the diploid).

Carry out a Dominance/Recessivity test:
Cross mutant with WT: diploid offspring need to show WT phenotype.

If carried out using dominant mutant phenotype, functional complementation assay will only give mutant and there will be no complementation, even if mutants are in different genes.

Question 3

What is a Complementation Assay?

Answer 3

Crossing 2 mutant haploids to create diploid cells, in order to ask whether diploid phenotype is WT or mutant.

If gives WT phenotype, the mutants complement and mutant genes are different.

If gives mutant phenotype, the mutants DO NOT complement and involve the same gene.

= Complement when each strain brings the active protein that other strain cannot produce.
= reciporcal contribution to pathway.