Principles of Genetic Recombination (With Diagram)!
Principle # 1. Transformation:
In this process only a limited amount of DNA is transferred from one strain of bacterium into the other in solution. It was first observed by Griffith (1928) in Pneumococcus pneumoniae (then called Diplococcus).
Evidence for this was obtained in the following experiment by him:
The Pneumococcus bacteria has both the virulent (micro-organism, capable to produce the disease) and non-virulent strains. Both strains differ in their morphology and colony characters. The virulent strains are capsulated i.e., have a capsule around the cell and form a smooth colony on growth medium. The avirulent strains are non-capsulated and form a rough colony.
Griffith injected mice subcutaneously with virulent strain bacteria. It caused death of mice. The avirulent were harmless to mice when injected. In the same manner heat killed virulent strain failed to kill the mice. However, when the living avirulent strain and the heat killed virulent strains were mixed and injected—the mice died. It means avirulent bacteria are changed into virulent bacteria.
Griffith isolated the bacteria from dead mice (Fig. 2) which had the characters of the virulent strain and proved virulence on injection into other mice. Later, Avery, Macleod and McCarty (1944) demonstrated this phenomenon in laboratory and found that the substance responsible for the transformation is DNA.
It was reported in many other bacteria such as Neisseria, Hemophilus etc. That part of DNA strand in which quality of transformation lies is called transfer factor and the cells in which transformation can be affected are known as competent cells.
These are important steps:
a. Adsorption of DNA:
A number of donor cells break apart and an explosive release and fragmentation of DNA takes place. A fragment of DNA, present in the medium gets adsorbed on the recipient cell. Its a double stranded DNA (Fig. 3 A). Single stranded DNA, however, neither penetrates nor inhibits the transformation.
b. Entry and integration of DNA:
The DNA fragment enters the bacterial cell (Fig. 3 B). Immediately it tends to become single stranded, thus forming two strands. One strand of this DNA molecule is integrated into the recipient cell, the other is broken, displaced and discarded (Fig. 3 B, C).
The end production of this recombination in duplex hybrid consisting of host DNA and the donor DNA. This results in the development of new phenotypic characters, which are exhibited in the progenies as stable and heritable character. The transformed chromosome replicates during binary fission (Fig. 3D).
Principle # 2. Conjugation:
In this method the parent cells come together in pairs (conjugate) and the genetic material of one is passed into the other generally by the formation of a bridge. It was first discovered by Lederberg and Tatum (1946) in Escherichia coli. In 1956 Woolman and Jacob described it in detail.
Evidence for this was obtained in the following experiment by them:
Out of two different strains, one parental strain (A− B−C+ D+ Sr Ps) requires factors A and B for growth and was resistant to streptomycin and sensitive to phage. The other parent required supply of growth factor C and D and was streptomycin sensitive and phage resistant (A+ B+ C−D−Ss Pr). Individually, both the strains were unable to grow on minimal media (a media which supplies cell all the minimal nutritional requirements is called a minimal medium).
But when the two strains were mixed and then spread on the same minimal medium they grew in the absence of any of the growth factors A, B, C or D. The growth of the colonies suggests the development of recombinant bacteria which must be A+B+C+D+ genetically.
When tested for streptomycin and phase resistance, these markers also appeared in new combination. None of the parents was double sensitive (Ss Ps or Sr Pr) or double resistant. But after the above experiment, the two colonies (3 and 4) were double resistant (Sr Pr) as they grew on both streptomycin and phage containing media. Colonies 6, 7, were double sensitive (Ss Ps) as they did not grow on any plant (Fig. 4).
Most of the experiments have been conducted with the specific strains of Escherichia coli particularly strain k12. Conjugating strains of E. coli show the sexual difference, one acting as donor of genes (male) and the other recipient of genes (the female). The transfer of the genetic material is unidirectional. The donor cell transfers parts of the genome (set of genes) to the recipient cell.
The cells of the opposite strain come together in pair. The donor cell has a special kind of pili known as sex pili, which helps to attach it to the cell wall of the recipient cell. Sex pili are having a hole of 2-5 mµ in diameter. DNA molecule passes through this passage in an uncoiled state. These sex pili are present only in donor cell, therefore, E. coli is sexually dimorphic.
The male bacterium also differs from the female in having the fertility factor called F. factor. The F. factor is an infective element and is now called as an episome (a term coined to denote genetic elements which can exist in two alternative states: lying free in the cytoplasm, or integrated with the bacterial chromosome).
If episome lies free in the cytoplasm it is called F factor, but if it is integrated with DNA or bacterial chromosome it is known as Hfr male (High fertility male or High frequency of recombination).
Now there are two different types of processes in conjugation depending upon whether the male cell is F or Hfr:
(1) In F+cells episome lies free in the cytoplasm. In conjugation between F+ and F−, the bacterial chromosome is not involved. The episome replicates and only one of the replication of the factor passes into the female cell. Thus, the receipent is coveretrd into the male cell and subsequently can conjugate with another unifected cell (fig. 5).
(2) In Hfr cell episome is integrated with bacterial chromosome. The bacterial chromosome breaks at the site of attachment (Fig. 6) and becomes a linear DNA molecule, having the F.factor always at the hinder most part. Chromosomal replication starts at the end which is directed towards the conjugation tube. DNA strand after replication transfer to F−cell. It takes about 100 minutes for a complete transfer of the genophore to occur.
Wollman and Jacob (1956) experimentally demonstrated that the first gene of the male starts entering the female cell in about 8 ½ minutes of making. This is followed by gene B at 18 minutes, gene C at 9 minutes, gene D at 11 minutes, gene E at 18 minutes, and gene F at 25 minutes.
But in nature the making never lasts so long. Due to interruption the transfer is usually not complete so that the resulting zygote is a partial zygote. The zygote cell (recombinant) shortly discards the fragment of the donor chromosome and reverts to the haploid state.
Thus bacterial conjugation differs from the sexual reproduction in two respects:
(i) Absence of meiosis and
(ii) Formation of a partly diploid zygote.
In addition to Escherichia, conjugation has also been observed to occur in strains of other bacteria such as Salmonella, Pseudomonas, Shigella and Vibrio etc. The conjugation may take place not only between strains of one and the same species, but between cells of different species which leads to the development of changes in strains known as inter-specific recombinants.
Principle # 3. Transduction:
In this process genetic material is transferred by means of a temperate phage. It was first discovered by Zinder and Lederberg (1952) in Salmonella typhimurium. Evidence for this is obtained in the following experiment conducted by them:
A U-tube was taken which had a sintered glass filter (deposited by silica by springs) between its two arms, through which bacteria could not pass (Fig. 7). Two auxotroph’s (nutritionally deficient strains) were grown in two arms, Salmonella strains (strain A) requiring tryptophan for growth were grown with Salmonella strains (strain B) requiring histidine for growth but susceptible to the bacteriophage of lysogenic Salmonella (strain A).
The medium from one arm could freely go into the other arm. Salmonella cells that require neither histidine nor tryptophan were produced, technically known as prototroph or mild forms as against auxotroph’s which are deficient in some enzymes (Fig. 7).
There are two types of growth cycles in bacteriophage (Fig. 8 A-C):
In lysogenic cycle the virus does not multiply and there is no death of host cells. Such bacteriophage is called temperate phage. The viral genome enters inside the bacterial cell and may exist outside the bacterial chromosome as F-factor or may attach to the bacterial chromosome (as F-factor does in Hfr strains).
The viral genome in this integrated sate is called pro-phage. The bacterium that carries the pro-phage is said to be lysogenic and the phenomenon where the bacterium and phage DNA coexist is called lysogeny. In test tube, strain A is a lysogenic strain. The phage does not cause the lysis of the bacterium. It may remain lysogenic for many generations during which time the viral DNA replicates together with the bacterium.
However, at some point in future, the phage stops coding repressor protein, and the lytic cycle begins. (In lytic cycle the sensitive bacterium lysis and large number of newly formed virus particles are liberated. Such bacteriophages are known as virulent or lytic phages).
The viral DNA which is attached to the bacterial genome, now breaks free and directs the synthesis of those proteins which are required for the formation of new viruses. In detaching, however, the viral DNA may carry with it a few bacterial genes from the bacterial chromosome. These genes are replicated along with the viral genome and become part of the new phage particles.
The lytic phages are very small and are able to pass through filter. They attach the salmonella (strain B) in the other arm which were sensitive to phage. The phage DNA enters the new bacterial cell and inserts onto a new bacterial chromosome. The bacterial cell now contains its own genes plus several from strain A. The host cell shows lysis and new phage particles are formed.
These phages accidently have a segment of the chromosome of strain B. Such phages are called the transducing phages. From here these transducing phages move through the sintered glass filter and attach the strain A where they behave as a normal temperate phage. The DNA of the strain B brought by the temperate phage integrates and replaces a homologous segment in strain A.
As a result a new (transduced) recombinant is formed. This type of transduction is called specialised transduction because specific genes are removed from the bacterial chromosome depending upon where the viral DNA was attached e.g., Lambda virus (Fig. 9).
There is another type of transduction. It is called generalized transduction. It is brought about by the plasmid i.e., by those bacterial segments which are not attached to bacterial genome e.g., PI phage. The phage DNA lies in the cytoplasm of bacterial cell and starts producing copies of it-self for new phage particles.
In this process some segments of bacterial chromosomes are incorporated into new phage particles while others have only phage DNA (Fig. 10). Phage particles are released after the lysis of the bacterial cell. Phage particles which contain segments of bacterial chromosome are capable to transduce bacterial cell. However, the particles with only phage DNA are incapable of transduction.
The incorporation of tiny fragments of bacterial chromosome with phage DNA is of rare occurrence. It is a random process and may involve any of the bacterial genes, hence named as generalized transduction (Fig. 10).
All the transduced genes do not get integrated into the host genome. When integration occurs with genome of recipient, the transduction is said to be complete transduction. Failure of the transduced gene to be associated with recipient DNA is termed as abortive transduction. The recipient after complex transduction is called recombinant.