Structure of Gene
A gene is neither a functional, nor a mutational or a recombinational unit, but it is a complex locus, whose fine structure should be studied. Such fine structure has been studied in a number of cases using higher resolving power of recombination technique. One may like to consider analogies, where a cell is a unit of life, but higher resolution through sophisticated microscopes discovered subcellular particles, and also where an atom is a unit of structure of an element, but subatomic particles like electrons, protons and neutrons are now known. By corollary, gene is a unit of heredity but its fine. structure has been studied through high resolution of the genetic systems. As an electron microscope gives higher resolution for the study of cell and its subcellular structures, a very big population in a genetic experiment will increase resolution of genetic system. Fine structure of a gene was not discovered early in the present century, because limited populations were used for recombination studies, which was unavoidable due to the nature of organisms used for study. When bigger populations were studied, intragenic recombination, whose frequency would be very low, could be discovered and a gene could be resolved into smaller units. This will be illustrated with the help of few examples.
Fine structure of lozenge locus in Drosophila
The results of Green and Green (1949) and Green (1961) on intragenic recombination in lozenge locus in Drosophila demonstrated that 13 alleles can be located on atleast four mutational sites separated by distances proportionate to recombination frequencies. The alleles on different mutational sites could be subjected to cis-trans test, and complementation relationships showed that all 13 alleles exhibit lack of complementation among each other, so that they belong to only one functional units.
Fine structure of rI locus in T4 phage
Distinction between wild type and rII mutants using K strain of E. coli-
The most refined analysis of a single gene ever conducted is the one undertaken by Seymour Benzer for a locus in T, bacteriophage infecting E. coli. This locus is known as rI locus and a mutant at this locus is responsible for the formation of rough plaques or colonies. This locus had largest number of rapid lysing (r) mutants, and is called r/Ilocus. It can be distinguished from other r loci, by the inability of rll mutants to produce plaques on lysogenic ‘K’ strain of E. coli which carries à prophage. The rII mutants, may though infect ‘K’ strain, but cannot cause lysis and are, therefore, unable to produce any plaques (plaque is a clear space on petri dish formed due to destruction of bacterial cells due to infection by phage and is characteristic of a phage). In contrast, these rII mutants make large sharp plaques on E. coli, strain B. The wild type phage T₁ (II) will make small and fuzzy plaques; both on B and K strains ,,Further, when ‘K’ was infected simultaneously by rll and rII, large plaques were formed, since rIIt helps in lysis so that rll may express. These distinguishing features enabled Benzer to distinguish mutants and wild type phages with high efficiency.
In order to find out complementation relations between different rII mutant alleles. Benzer used two different rll mutants, arbitrarily designated as rIP and rIP. He allowed mixed infection of K strain by these two mutants. Although in most cases, this does not result into lysis and plaque formation, in some cases it does lead to plaque formation. If two mutants did not form plaques on mixed infection, they were placed in the same group, but if plaques were produced, the two mutants involved in mixed infection were placed in two different groups. In this manner, two groups A and B could be established in rll region. All mutants with the help of complementation test could be classified in these two groups, in such a manner that two mutants from group A or two mutants from group B could not cause plaque formation but mixed infection by one mutant of group A and another of group B, could cause plaque formation. Since groups A and B are distinguished on the basis of cis-trans test, these were termed as cistron A and cistron B. Mutants belonging c to the same cistron i.e. A or B would exhibit cis-trans phenomenon, meaning that they would give wild type only in cis configuration and not in trans configuration. Two mutants from different cistrons (A-and B) would give wild type (plaque formation) even in trans configuration, which in other words is called complementation. For the purpose of cis configuration, a chromosome with two mutants can be obtained through mixed infection followed by recombination.
From the complementation test, it is obvious that in rII region, two cistrons A and B are independent functionally and must be responsible for sequential synthesis of two separate products, which presumably are polypeptide chains. Therefore, all mutants belonging to one cistron share a common deficiency, which is different from the deficiency due to mutants belonging to the second cistron. When two mutants belong to same cistron, both are deficient for same product and therefore, they cannot complement, but when two mutants belong to two different cistrons, they, being deficient for different products, can complement, and may express wild phenotype i.e. lysis and plaque formation.
Once rII mutants were classified in cistron A and cistron B, Benzer was interested in analysing mutants, belonging to same cistron. It was, therefore, necessary to subject them to recombination test to find out whether they are located on same site or different sites separable by recombination. For this purpose, Benzer classified all mutants into two categories (i) revertant type- point mutations and (ii) non-revertant type- deletion mutations (multisite mutations). For recombination analysis, to reduce labour, Benzer made use of non-revertant mutants which were due to deletions (that these were deletions was obvious from several tests including their non-revertant nature). These deletions were arranged insets of overlapping deletions representing segments of different sizes in rII region. The principle involved in this technique was that if a particular point mutant lies in the region of a deletion represented by a rII mutant, then on mixed infection with this deletion mutant, the point mutant will not be able to give rise to a wild Using non-revertant (deletion) mutants having successive overlapping deletions of smaller lengths, one could locate a mutant to a fairly small region. All point mutants located in this particular small segment of rII region, could, then be subjected to recombination test. For this purpose, two mutants at a time were used for mixed infection of E. coli B strain and the lysate produced on plaques formed on B strain was used for infection on K strain to find out the frequency of wild type phage particles produced, because this will represent the recombination frequency. By this method, Benzer was successful in estimating recombination frequencies as low as.0001 per cent, although later, frequencies as low as.00001 per cent could be estimated. Thus Benzer was not only able to divide rII region into cistrons A and B, but was able to classify mutants belonging to same cistron into hundreds of mutational sites separable due to recombination.
Through recombination analysis, about 2000 mutations were mapped in rll region and were found by Benzer to occupy 308 different sites called mutational sites. These sites were, although scattered fairly evenly throughout the length, some of them were called hot spots since they had numerous mutations relative to other sites. In contrast, some sites had no mutants and were, therefore, called zero sites. Benzer eventually estimated 400-500 mutational sites in rll region and called each of them a unit of mutation or a muton.
- Concept of Allelomorphism | Position Effect
- Structure of Chromosome | Euchromatin & Heterochromatin
- Molecular Structure of Chromosome (Nucleosome Structure)
- Stages of Mitosis (Prophase, Anaphase, Metaphase & Telophase)
- Stages of Meiosis (Cell Division)
- Synaptonemal Complex- Structure, Function, Significance etc.
- Structure of Nucleus
- Structure of Nucleolus
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