In this article we will discuss about:- 1. Discovery of Transformation 2. Process of Transformation 3. Competence 4. Linkage and Gene Mapping.
Discovery of Transformation:
The phenomenon was first discovered in 1928 from Griffiths experiment with Diplococcus pneumoniae. As this historical experiment has been instrumental for identification of DNA as a hereditary material. Briefly, when a suspension containing a mixture of heat-killed virulent, encapsulated cells and live, non-virulent, non-encapsulated cells was injected into a mouse, a small fraction of the live bacteria became transformed into the virulent encapsulated type.
The transforming ability was inherited by the descendants of the newly transformed live strain. It was inferred that when cells of the virulent strain are killed by heat, their chromosomal material which is somehow liberated from heat-killed cells can pass through the cell wall of the living cells and become incorporated in the host chromosome. Although this experiment involved genes that control presence/absence of the capsule, later on genes controlling other characters could also be transformed by addition of chromosome fragments.
Transformation has proved useful in locating genes in bacteria like Bacillus subtilis, E. coli, Hemophilus influenzae, Rhizobium, Neisseria and others. Attempts have been made to find out if transformation occurs in higher organisms including mammals and man.
All results have been so far negative, except for some special cases where viral chromosome, for example of SV40 (simian virus), which can transform normal human cells in culture into cancerous ones. The mechanism of transformation is not fully known.
Process of Transformation:
When recipient cells are grown in presence of killed donor cells, transformation is observed. The DNA of donor cells is transferred to recipient cells where it undergoes genetic exchange with recipient chromosomes to produce recombinant progeny.
Analysis of the process indicates that successful transformation depends upon several factors: size of donor DNA fragments which varies in different species of bacteria; molecular configuration of donor DNA which must be double stranded; physiologically competent state of recipient cells which occurs over a limited period in the growth of a culture and the ability to achieve this state is an inherited character; the amount of DNA added per recipient cell, i.e., the frequency of transformation increases with the concentration of DNA up to the point where 10 molecules of DNA per cell are present. Further increase in concentration of DNA seems to have no effect.
Competence of Transformation:
When a recipient cell is able to absorb donor DNA and become transformed, it is said to be a competent cell. The development of the competent state appears to be related with cell density. Thus most cells growing in culture become competent when a critical number of cells is attained. Competence therefore, represents a transient phase in the life of a population. Its time of occurrence and duration are characteristic for a bacterial genus.
As competence is acquired by cells in culture, a protein called competence factor is produced which confers competence on other cells. This factor seems to act by changing the cell surface properties either by formation of receptor sites, or increased permeability to donor molecules. Cyclic AMP is also found to play a role in the development of competence. When added to the medium, this compound greatly increases the level of competence among the cells.
Uptake of DNA:
The double stranded donor DNA molecules bind to the receptor sites on the recipient cell surface. Both homologous DNA and DNA from an unrelated species will be taken up by Pneumococcus whereas Hemophilus will take up only homologous DNA. The donor fragments are cleaved by endonuclease on the surface of the recipient cell to a size which varies in different bacterial species.
After attachment to the recipient cell wall, the donor DNA is actively transported inside the cell. Soon after uptake, one strand of the donor DNA fragment is degraded so that it becomes single stranded (Fig. 17.1). Immediately there is no transforming activity (eclipse period).
Eventually the fragment pairs with that region of the recipient cell chromosome with which it is homologous. Genetic exchange takes place and a single strand of donor DNA carrying one or more genes from the donor cell becomes integrated in the homologous portion (having corresponding sequence) of recipient DNA. The single stranded segment which breaks of from the recipient DNA is degraded in the cell and lost.
That transformation is a reversible process can be demonstrated experimentally. If donor DNA fragment contains a hypothetical gene t– and the recipient t+, the transformed bacteria are found to contain t–. When these t– bacteria are used as recipients for donor t+ DNA, the resulting bacteria become t+ again.
Studies with Bacillus subtilis have shown that when DNA from an animal virus or bacteriophage is used in transformation, intact virus particles are formed inside the recipient bacterial cell. The process is called transfection. In this case there is no need for donor DNA to become integrated into the host chromosome.
When such a bacterium comes in contact with the animal host which the virus is able to infect, it releases the contained virus particles causing infection of the host animal. Experimentally transfection can be assayed by formation of plaques when infected bacteria burst to release the virus progeny.
Linkage and Gene Mapping by Transformation:
Fragments of donor DNA which are involved in transformation can be used for detecting linkage and gene order in bacteria. The method consists of counting the number of double transformants (that is cells transformed for two genes) as well as single transformants produced by a single gene.
Suppose two genes E and F are placed distantly apart on the bacterial chromosome. The probability of both occurring together in the same fragment and producing double transformants is quite low. But a cell can become doubly transformed if it receives two separate donor fragments, one carrying E, the other F.
The probability for such an event would equal the product of their separate probabilities in producing single transformants for E and for F and would be lower than the single events.
But if E and F genes are closely linked, the probability that both are present on the same fragment and produce double transformants is high. When the experiment is performed, the number of single and double transformants will also depend upon the concentration of DNA containing donor fragments that are given to the recipient cells.
A graph can be plotted to illustrate the curves for single and double transformants with decreasing concentrations of transforming DNA. If genes E and F are linked, the curve for double transformants for E and F must be similar to the curve for single transformants for E and for F.
By using larger fragments of donor DNA it is possible to map gene loci in the vicinity of E and F, and also other genes in the genome. Three gene mapping, similar to the three points cross in higher organisms is also done for mapping genes in bacteria.