In vitro complementation with mutants

BIOC/MCB 568 -- Fall 2010
John W. Little--University of Arizona

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We discussed in class the approach of in vitro complementation, which might also be termed fractionation-and-reconstitution, or, more informally, divide-and-conquer. This approach can be adapted for use with mutants.

The crucial difference between using mutants and the usual in vitro complementation is that the goal of the mutant approach is to identify the gene product of a particular gene for which you have a mutant, while in the in vitro complementation approach you are trying to identify all the components. There's no guarantee that the mutant approach will give you an interesting component (but then there aren't many guarantees in research anyway!).

There are several stages in this process, which are first listed then explained.

1. Isolate mutations affecting the process you are interested in studying.

2. Carry out "complementation tests" to divide your mutations into ones affecting different genes. E.g. suppose you have three mutations, A, B, and C. If A and B are in the same gene, there's no sense in giving that gene two different names; the mutations would be two different alleles of the same gene. This procedure identifies a set of genes affecting the process you're studying. You give these names that hopefully reflect the process you're studying, e.g., dnaB.

3. Make extracts of the mutant strain; suppose it carries a ts mutation in dnaB. (Recall that you need conditional mutants to study essential functions like DnaB). Assume that life is kind and that the only defect in this extract is a non-functional DnaB protein. In your in vitro assay for your process (DNA replication in the example), this extract will be inactive for that reason and only that reason.

4. Fractionate the wild-type extract (as in the in vitro complementation approach), add fractions to the dnaB extract, and save the fractions that restore activity to that inactive extract. Keep purifying until you have a pure protein. In principle, this should give you a preparation of DnaB protein, purified away from everything else not needed for activity.

Further explanation

1. It's not important for our purposes how mutants are isolated, but here's a little more detail. In the case discussed in class, ts mutations affecting replication, these were isolated in two different ways. The first was to assemble a very large collection of ts mutants (ones that can grow at the permissive temperature but not at the restrictive temperature), and screen through these for those that can't make DNA at the restrictive temperature. The second was to isolate mutants that couldn't make DNA by making DNA synthesis lethal (for the aficionados, this was done by allowing replication to occur in the presence of the thymine analogue 5-bromouracil, which gets incorporated into DNA, followed by irradiation of the cells with UV light; DNA containing 5-BU is destroyed, so only cells that didn't incorporate 5-BU survive).

In either case, one winds up with a collection of mutants that can't make DNA (in this case) at the restrictive temperature.

2. Take two mutations and ask if they affect the same gene. This is easiest to envision in cells that are diploid (like diploid yeast or humans). Make a cell with one copy of the chromosome from each of the two mutants. If the two mutations affect the same gene, then function will not be restored; there's no good copy of the gene. If they are in different genes, for each gene there will be a wild-type copy from the other parent, and function will be restored. This allows one to divide the mutants into "complementation groups", which usually correspond to genes. You then give these genes names such as dnaA, dnaB, dnaC, etc. These names eventually wound up being given to the proteins, at least in some cases, such as DnaB helicase. Thought question: Would this "complementation test" work if the mutations are dominant?

3. Self-explanatory

4. This works the same way as the in vitro complementation.

One pitfall of this approach is that you may not have mutants affecting all the components of a process.

Another is that it is possible that, in your mutant extract, several components may be damaged or missing. For instance, if a protein is part of a complex, it's possible that in the mutant extract the lack of one component will destabilize other components of the complex. We'll see an example of this when we talk about eukaryotic transcription.