Viruses replicating themselves in the bacterial cells are known as phages. They are capable of surviving through a different mechanism. Their genes are easy to study since these entities are totally dependent on the bacteria. Plus they naturally exhibit the property of recombination of genetic material. Thus bacteriophage gene mapping is an interesting topic for geneticists. These phages are vectors since they are capable of transferring the genetic material from one bacterial cell to another bacterial cell. They are model organisms used in biological research. A gene map is a linear designation of mutant sites within a gene based on intragenic recombination. It could exhibit a sequence with regulatory sites, introns, exons and other elements. The principles used in eukaryotic gene mapping are applicable for mapping phage genes. Two main principles follow this. The genetic material gets exchanged between strains differing in the genetic markers. The second principle involves the detection of recombinants and counting them.
The intragenic or intergenic recombinants are mapped separately. These methods involve mapping the mutation sites. The unit of mutation and recombination is a DNA base pair. Mapping of bacteriophage genes requires the knowledge of the phenotypes. Several mutations give differences in plaque appearance. Consider two phage strains differing in genotypes. One phage has a genotype h+r. It is wild-type for a host range. The mutants have an hr+ genotype. The phage with a mutant allele produces a clear plaque. The phages with the wild-type allele produce cloudy plaques. A genetic cross is performed to map these two genes. Two E. coli strains are strain B and B/2. First, the strain B bacteria are coinfected with two bacteriophages named h+r and hr+. The phage chromosomes replicate within the bacterial cells and undergo recombination. Then the cell lysis releases the progeny phages along with the recombinant phages.
Fine structure and deletion mapping techniques were done using bacteriophage genes. The review focuses on both the mapping techniques.
Fine structure analysis:
Image 1: Fine structure mapping
Mutation studies in Drosophila led to the conclusion that the gene was sub-divisible by mutation and recombination. C.P. Oliver studied and proposed this concept in 1940
Intergenic mapping involves mapping the distance between mutational sites in different genes. Intragenic mapping involves mapping mutational sites within the same gene. Seymour Benzer worked on fine details of phage T4 genes. Fine structure mapping involved details within the gene. He worked on rII mutants of T4 phages. The rII mutants possess two main properties such as distinct plaque morphology and host range properties. When the r+ wild-type phages infected the E. coli bacteria plated on a solid medium, small turbid plaques with fuzzy edges appeared. The rII mutants produced large and clear plaques. There is a difference between wild-type and mutant strains. Wild-type r+ strains grow and lyse strain B or K12(λ). The rII mutants grow only on strain B. Thus the experimenter set out to construct a fine structure map of rII region using E. coli strain B as the permissive host. The experiment involved 60 independently isolated rII mutants. Different mutations formed the basis of crosses.
The rIIx and rIIy mutants were allowed to cross. Different mutations were written x and y respectively. Four types of progeny were the parental type such as rIIx, rIIy, four double mutants rIIx,y with r+ wild-type. The relative frequencies of the parental and the recombinants depended on the distance between the two alleles. The phage progeny were plated on E. coli B strain to calculate the total number of phages per milliliter.
Recombination frequency for two alleles
= (2 x No. of r+ recombinants x 100%)/ Total number of progeny
The number 2 indicates the double recombinants. The chances of reversions were lower than the smallest recombination frequency. Homoallelic and heteroallelic mutations were involved. Homoallelic mutations involved changes in the same nucleotide base pair within a gene. Heteroallelic mutations involved changes in different nucleotide pairs. The phage T4 gene map is about 1500 map units.
Deletion mapping was used to localize unknown mutations. Maximum mutants isolated from the experiment were known as point mutants. The point mutations can revert. However, some rII mutants did not revert. Nor did they produce any recombinants. Such mutants were known as deletion mutants. Such mutants had lost a segment of DNA. The crosses performed between the mutants were analyzed. Seven standard deletion mutants involved in the cross. A1 to A6 and segment B included a total of seven segments. Thus, deletion mapping involves overlapping deletions to localize the position of an unknown gene on a chromosome or a linkage map. Gene structure analysis and mapping revealed that genes are units of mutations, recombination, and function. Determining the number of genes involved a test known as complementation test. The gene mapping studies and fine structure analysis revealed that the unit of mutation and recombination are the same. The base pair in DNA is the main criteria for determining both the factors.
Complementation test:
Image 2: Complementation test: The first Petri-plate shows the plaque formation. The second Petri-plate has no plaques. Thus, it helps in finding the complementation.
The test was used to find out whether the two different mutants belonged to the same gene. It is a mating test that determines whether the two different recessive mutations on opposite chromosomes of a diploid or a partial diploid will not complement each other or have a mutant phenotype. However, the same two recessive mutations on the same chromosome may show wild-type phenotype. Thus, complementation test is a test for allelism. Let us consider two cases for our understanding.
In case I, bacterium E. coli K12(λ) is infected with two phages with rIIA mutation and rIIB mutation respectively. Both the mutants make non-functional A and B products respectively. In this case, complementation occurs because rIIA mutant makes functional B product and rIIB mutant makes functional A product. Thus, progeny phages result in the formation of plaques.
In case II, bacterium E. coli K12 (λ) is infected with two phages having mutations in the rIIA gene. Hence, a non-functional A product and a functional B product were synthesized. In this case, complementation does not occur. Phage propagation does not occur due to lack of functional A product. Hence no progeny phages are produced. Thus, no plaques were visible on the bacterial lawn. These studies are inclusive of fine structure mapping and deletion mapping. A complementation map is a diagrammatic representation of the complementation pattern of mutants. Two types of lines drawn in a complementation map include overlapping and non-overlapping lines. The overlapping lines indicate non-complementary mutants whereas non-overlapping lines represent mutually complementing mutants. A complementation map is linear and may show lesions.
References:
[1] Microbial Genetics, Keya Chaudhari
[2] Molecular Genetics of Bacteria, Jeremy W. Dale, Simon F. Park
[3] Genetics, G. Ivor Hickey
[4] IGenetics, Peter Russell
[5] Genomes, T.A. Brown
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