In this article we will discuss about the mapping of rII locus in T4.
The wild type T4 phage produces small plaques with rough edges on both strains B and K of E. coli. The r mutants (rapid lysis) are easily distinguished by their large, sharp-edged plaques. The wild type phage do not lyse and release progeny phage as rapidly as the r mutants. The r mutants are classified into groups depending upon their behaviour in E. coli strains other than B.
The rII group of mutants analysed by Benzer differ from the wild type as they do not produce plaques on strain E. coli K which carries phage λ (lysogenic for λ). The wild type T4 (rII+) grows on E. coli K (λ).
Thus an rII mutant produces r-type plaques on strain B of E. coli, wild type plaques on strain K12S (designated strain S) of E. coli, (that is one which does not harbour A,) and it forms no plaques on strain K(λ). The wild type T4 produces similar plaques on all the 3 strains B, S and K.
The rII mutants proved favourable for this study because it is possible to detect even a very small number of wild type particles among a very large group of mutants. The r group of mutants produces a distinct plaque type on strain B, and rII mutants are identified by testing on strain K.
On strain K(λ) only wild type will grow. Thus when the progeny of a genetic cross between two different rII mutants are added to E. coli K (λ) only the wild-type recombinants will form plaques and will be detected even when their frequency is as low as one per 106 progeny.
The cis-trans complementation tests first devised by Lewis for studying the gene in the lower eukaryotes were applied by Benzer to phage. He crossed pairs of rII mutants and found that they belonged to two functional groups, due to presence of two separate segments rII A and rII B in the rII region of phage chromosome. The two groups of rll mutants, namely rII A and rII B can be distinguished from their behaviour after mixed infection of strain K (λ).
When mixed infection is done using two different mutants one belonging to rII A, the other to rII B group, the two phages multiply, cause lysis of host cell and plaques are formed.
This means that normal polypeptides of both mutants are required to produce plaques—that is to say, the two complement each other (Fig. 22.2). But if the two mutants used for mixed infection of K(λ) both come from the same group, either rII A or rII B, they will not be able to form plaques (non-complementing).
The fact that segments A and B show complementation demonstrates that A and B are independent, separate units. Each performs a different function resulting in distinct polypeptide end products. Polypeptides from both segments A and B are necessary for phage multiplication in strain K (λ). Thus A and B are two separate genes or cistrons (the term cistron was coined by Benzer).
The entire rII region is a single functional unit in the sense that all mutations within this region (both segments A and B) produce the rII phenotype. The polypeptide product of A is not fully known; however B codes for a cell membrane protein.
The following procedure was used by Benzer for mapping the rII region. Strain B cells were infected with a 1:1 mixture of two rII mutants. After cell lysis, the progeny recovered consisted of the two parental types and recombinants of two types—double mutants and wild type.
When progeny from many cells was considered, the two recombinant types were found in equal proportions. The progeny phages were made to infect K (λ) cells and B cells. The double rll mutants will not form plaques on K (λ), only the wild recombinant type will. Thus the total number of recombinants in the progeny are obtained simply by doubling the wild type plaques on K (λ).
On strain B all types of progeny, parentals and recombinants will form plaques. The proportion of parentals to recombinants are thus determined. In this way (Fig. 22.3) by crossing rII mutants in pairs, Benzer was able to map a large number of mutations in rII region.
The method described above is sensitive enough to detect one wild type phage in a million particles. The lowest frequency of recombination between two loci is an index of the smallest distance separating two loci within which recombination could occur. But r mutants can revert to the wild type as first found by Hershey.
Thus although Benzer had expected to detect a recombination frequency as low as 10-6, the lowest he actually found was 10-4 (0.01 %). Since this represents half of the recombinant types, the lowest frequency would be 0.02 %.
Therefore, the lowest frequency of recombination that can be accurately determined between two rII mutants is 0.02 per cent (that is a distance of 0.02 map units). Now the entire circular genetic map of T4 is 1500 map units containing about 2 x 10-5 nucleotide pairs.
Therefore a map distance of 0.02 map units would proportionately be 1.3 x 10-5 of the entire phage genome. Thus the minimum distance between two points within which recombination can occur would be (1.3 x 10-5) x (2 x 105) which amounts to about 3 nucleotide pairs. Later work on phage DNA by Yanofsky has shown that recombination can occur even between adjacent nucleotides.
With this technique Benzer mapped about 2000 mutations in rII region. Obviously it became difficult to cross such a large number of mutants in mixed infections. Fortunately there was a second class of mutations called deletion mutations which had a deficiency in either the A or B segment.
In phage deletions are identified by their stability and their failure to have second mutations which revert them to the wild type. Further, recombination does not occur in the region of the deletion.
In deletion mapping a phage with a deleted segment and another phage carrying a mutation at the identical site as the deletion are used in mixed infection of strain E. coli B cells. Their progeny is plated on K(λ).
If the deletion and the known mutation occupy the same site, then no wild type recombinants will occur. But if the sites are different then wild type recombinants would appear (Fig. 22.4). By testing a deletion with a number of known mutations, the length of a deleted segment can be determined.
In this way a series of deletions showing gradations in length can be mapped. They are then used to map an unknown rII mutation by crossing a mutant with a series of deletions and observing whether recombination occurs or not (Fig. 22.5). The method involves selecting a group of deletion mutants whose lengths are known and crossing an unknown mutant with each one of them.
As shown in the hypothetical Fig. 22.5, crosses between the unknown mutant M and deletion mutants d2, d3, d4 and d6, no recombinants are recovered because the mutant site lies opposite a deleted segment of the second phage DNA.
But when the unknown mutant M is crossed with d 1, d5 and dl, recombinants are produced. In this way we can map the exact location of mutant M. The length of the DNA in rIIA gene is calculated to be 6 map units (800 base pairs) and in rII B region 4 map units (500 base pairs).
In complementation studies when each of two mutations impairs the same function, the mutants do not complement each other. But sometimes there is intragenic or interallelic complementation between the different mutant alleles of the same gene, due to which a small proportion of normal phenotypes is produced.
In some fungi and bacteria each of the alleles which are functionally related specifics the amino acid sequence of a different subunit of a single enzyme. Each subunit performs its own function.
The association of subunits produces the secondary structure of the polypeptide; this secondary structure is responsible for enzyme activity. Thus intragenic complementation is observed through the secondary structure of a polypeptide, and not its primary structure which is determined by the amino acid sequence.