You do a second mutant hunt to screen for histidine mutants and isolate 15 mutan

Question

You do a second mutant hunt to screen for histidine mutants and isolate 15 mutants. You find that the his- phenotype is recessive in mutants 1-14, but dominant in mutant 15. You then organize the mutants based on complementation groups a) Describe the experiment that you performed and the results that you obtained which allowed you to determine that the his-phenotype is recessive in mutants 1-14, but dominant in mutant 15 b) Describe how you would determine complementation groups for each of your mutants and explain what it means if two mutant are in the same complementation group as opposed to in different complementation groups c) The following is the result of a complementation assay. Here (-) represent no growth on minimal media and (+) represent growth on minimal media. Based on the information provided, arrange the mutants into complementation groups. A1. A2 A3 A4 A5 A7 A8 A9 A10

Explanation / Answer

Mutants which fail to complement each other fall in a complementation group.

Complementation test is performed to determine whether the mutations are on same gene or in different genes.

a)The experiment we can perform involves studying the biosynthetic pathway of histidine.

Histidine biosynthesis involves a cascade of steps mediated by different enzymes coded by different genes. Product of one gene determines the next step in the pathway.

So the mutants which have mutation in gene X will cannot synthesize histidine . Those mutants require histidine for their growth.

+ is prototrophs , growing in absence of histidine(minimal media)

- is auxotrophs , requires histidine for growth(MM)

When mutant A1 is mated with itself, the resultant diploid is still remain auxotroph because it is homozygous for the defective allele.

When mutant A1 is mated with A2, then mutants complemented , and the prototrphy restoration occured.

Likewise for other mutants as well.

In this way we can score which his-phenotype is recessive in mutant 1-14.

b) If a mutant having mutation in gene X coding for an enzyme A and other mutant has mutation in gene Y coding for enzyme B. Since both the genes are required for the histidine biosynthesis pathway .Neither of them can make histidine alone but when mutations are combined then one chromosome have normal copy of gene X will make ENZYME A, while other mutant can synthesize ENZYME 2, In this way both can together form a wild type phenotype(prototrophy).They are called to be in different complementation

When mutant A1 is mated with itself, the resultant diploid is still remain auxotroph because it is homozygous for the defective allele., they are called in same complementation group.

c ) Likewise mutants which fail to complement each other will be categorised in one group/same group

group 1 A1, A3, A5

group 2 A2, A4, A6

group 3 A7, A8

gruop 4 A9, A10

thus total number of complementation groups are 4

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