Cis-Trans Test: Complementation
- This was studied in Bacteriophage Genetics, where mutation of a gene designated r, for “rapid lysis was examined.”
- It turned out that actually there are three different gene loci – rI, rII, and rIII – mutations in any one of which produced a rapid-lysis phenotype.
- But, in addition, there were many mutations found in each of these. Could wild-type virus be formed by recombination between mutations within the same gene?
- Seymour Benzer decided to find out. In Bacteriophage Genetics, the recombination frequency between different genes is low (on the order of 10-2).
- One would expect that recombination frequencies between mutations in a single gene would be far lower (10-4 or less).
- Fortunately Benzer could exploit a phenomenon to enable him to detect such rare events: rII mutants can infect – but not complete their life cycle in – a strain of E. coli designated K.
- Wild-type T4 can complete its life cycle in both strains.
- The procedure was to infect strain B (Figure 2.23) in liquid culture with two mutants to be tested (designated here as rx and ry).
- After incubation, these were plated on a lawn of:
- strain B — which supports the growth of all viruses thus giving the total number of viruses liberated.
- strain K — on which only wild-type viruses can grow (Figure 2.24).
- The recombination frequency between any pair of mutations is calculated as Recombination Frequency = 2 × number of wild-type plaques (strain K plaques) ÷ total number of plaques (on strain B).
- You have to double the number found on strain K because you only see one-half the recombinants — the other half consists of double mutants.
- Using this technique, Benzer eventually found some 2000 different mutations in the rII gene.
- The recombination frequency between some pairs of these was as low as 0.02.
- The T4 genome has 160,000 base pairs of DNA extending over ~1,600 centimorgans (cM).
- So 1 cM = 100 base pairs
- So 0.02 cM represents a pair of adjacent nucleotides.
- From these data, Benzer concluded that the
- Smallest unit of mutation and
- The smallest unit of recombination was a single base pair of DNA.
In other words,
- These mutations represent a change in a single base pair – we call these point mutations.
- Recombination between two molecules of DNA can occur at any pair of nucleotides.
- As we saw above, rapid lysis (r) mutants were found that mapped to three different regions of the T4 genome: rI, rII, and rIII.
This meant that
- Those in different regions were not alleles of the same gene.
- More than one gene product participated in the lysis function.
Even within one “locus”, rII, there turned out to be two different stretches of DNA both of which were needed intact for the lysis function. This was revealed by the complementation test that Benzer used. In this test,
- E. coli strain K (which rII mutants can infect but not complete their life cycle) – growing in liquid culture – was
- Co-infected with two different rII mutants (here shown as “1” and “2”).
Note that this procedure differs from the earlier one (recombination) in that the nonpermissive E. coli K is used for the initial infection (not strain B as before). Neither strain rII“1” nor strain rII“2” is able to grown in E. coli K. But if the lost function in rII“1” is NOT the same as the lost function in rII“2”, then each should be able to produce the gene product missing in the other – complementation – and
- living phages will be produced. (Again, there is no need to count plaques; simply see if they are formed or not.)
From these results, you can deduce that these 5 rII mutants fall into two different complementation groups, which Benzer designated
- A (containing strains 1, 2, and 4) and
- B (containing strains 3 and 5)
Later work showed that the function of rII depended on the polypeptide products encoded by two adjacent regions (A and B) of rII (perhaps acting as a heterodimer). In terms of function, then, both A and B qualify as independent genes. In co-infections by two mutant strains,
- If either A or B is mutated on the same DNA molecule (“cis”), there is no function while
- If A is mutated in one DNA molecule and B in the other (“trans”), function is restored.
Complementation, then, is the ability of two different mutations to restore wild-type function when
- They are in the “trans” (on different DNA molecules)
- But not when they are in “cis” (on the same DNA molecule).
Benzer coined the term cistron for these genetic units of function. But today, we simply modify earlier concepts of the “gene” to fit this operational definition.