Genetic recombinations

GENETIC RECOMBINATION

Genetic recombination involves the physical exchange of genetic material between organisms. It involves the genetic exchange between homologous DNA sequences form two different organisms. Hence, it is also referred as homologous recombination. In classical genetics, it is referred as crossing over. In prokaryotes, RecA protein is a DNA binding protein involved in the genetic recombination. The following sequence of steps involved in the genetic recombination of prokaryotes.

Molecular mechanism of genetic recombination

The RecA protein is the key to homologous recombination. RecA is essential in nearly every homologous recombination pathway. The molecular mechanism of homologous recombination as event-wise as follows:

An enzyme that cuts DNA in the middle of a strand, known as an endonuclease, begins the process by nicking one strand of the first DNA molecule.
This nicked strand is separated from the other strand by proteins with helicase activity.
Single-strand binding protein then binds to the resulting ssDNA and the RecA protein binds to it.
This complex promotes base pairing with the complementary sequence in the second DNA molecule.
This in turn displaces the other strand of the second DNA molecule and is therefore called strand invasion.
The base pairing of one strand from each of the two DNA molecules over long stretches generates recombination intermediates, where each strand has originated from a different chromosome which is referred as heteroduplex region.
Finally, the linked molecules are separated or “resolved” by resolvases that cut and rejoin the second (previously unbroken) strands of both original DNA molecules.

These videos describe how recombination (cross over) occur. It may be for eukaryotic or prokaryotic cell

Three mechanisms of Gene Transfer

As discussed earlier, transfer of DNA from donor to recipient is the first step of homologous recombination. Three mechanisms of genetic exchange are known in prokaryotes:

(1) transformation, in which free DNA released from one cell is taken up by another;

(2) transduction, in which DNA transfer is mediated by a virus; and (3) conjugation, in which DNA transfer involves cell-to-cell contact and a conjugative (F) plasmid in the donor cell. The two DNA molecules from donor and recipient brought together in three different ways but after this, the homologous recombination is equivalent in all the cases.

In prokaryotes, only part of a chromosome is transferred; therefore, if recombination does not occur, the DNA fragment will be lost because it cannot replicate independently. Thus, in prokaryotes, transfer is just the first step in generating recombinant organisms.

A. Transformation

Transformation is a genetic transfer process by which free DNA is incorporated into a recipient cell and brings about genetic change. Several prokaryotes are naturally transformable, including certain species of both gram-negative and gram-positive Bacteria and also some species of Archaea. As the bacterial DNA is a large single molecule, when the cell is gently lysed, the DNA pours out. Because of their extreme length (1700 µm in Bacillus subtilis), bacterial chromosomes break easily and serve as source DNA for transformation. The average size of DNA as transformable element is about 10 kbp. A single cell usually incorporates only one or a few DNA fragments, so only a small proportion of the genes of one cell can be transferred to another by a single transformation event.

The ability of an organism to take up the DNA molecule is referred as Competence and the organism is said to be Competent. The competence of organisms varies among the organisms. Bacillus has 20% competence cells and E. coli and Streptococcus, 100% of cell become competence. The gram-ve bacterium, Haemophilus takes only double strand DNA whereas, the Streptococcus and Bacillus can take only single strand DNA, while the complementary simultaneously degraded.

The naked DNA first bound on the surface of the recipient cell with the help of surface proteins. A single strand of DNA is allowed to enter inside the cell. The entered DNA will be moved to chromosomal DNA and genetic recombination takes place with the help of RecA protein.

Griffith’s Experiment:

The discovery of transformation was one of the key events in biology, as it led to experiments demonstrating that DNA was the genetic material. This discovery became a cornerstone of molecular biology and modern genetics. The British scientist Frederick Griffith obtained the first evidence of bacterial transformation in the late 1920s. Streptococcus pneumoniae (pneumococcus), invade the body of mammals due to their presence of a polysaccharide capsule and cause disease. Mutants that lack this capsule cannot cause disease. Such mutants are called R strains because their colonies appear rough on agar, in contrast to the smooth appearance of encapsulated strains, called S strains. A mouse infected with only a few cells of an S strain succumbs in a day or two to a massive pneumococcus infection. By contrast, even large numbers of R cells do not cause death when injected. Griffith showed that if heat-killed S cells were injected along with living R cells, the mouse developed a fatal infection and the bacteria isolated from the dead mouse were of the S type. Because the S cells isolated in such an experiment always had the capsule type of the heat-killed S cells, Griffith concluded that the R cells had been transformed into a new type. This process set the stage for the discovery of DNA. (Refer lecture 2 and related video)

Artificial transformation:

The molecular biology techniques now used to transfer any naked DNA (in the form of vectors) to recipient cells are artificially.

Calcium chloride mediated transformation: The CaCl₂ washed recipient cells mixed with DNA allowed a temperature shock (42°C for 40-60 sec.) will allow the naked DNA to get transformed.
Gene gun (Particle gun): will allow transformation of plant tissues, yeast, algae etc.
Electroporation: Transformation takes place by means of electrical pulses.

Transformation in bacteria in natural conditions

B. Transduction

A DNA transferred from one cell to another through the agency of virus is referred as transduction. The transduction of bacteria occurs through bacteriophages. Some phages are lytic, which kill the host cell during replication, called lytic cycle and lytic phages. Ex. T2 phage; some phages replicate in the bacteria without killing the host cell, called as lysogenic cycle and temperate phage. The host cell is called as lysogen. Based on this behavior of virus, transduction occurs in two way. 1. Generalized transduction and 2.Specialized transduction.

Generalized Transduction

In generalized transduction, virtually any gene on the donor chromosome can be transferred to the recipient. Generalized transduction was first discovered and extensively studied in the bacterium Salmonella enterica with phage P22 and has also been studied with phage P1 in Escherichia coli. Zinder & Lederberg (1952) first identified generalized transduction in Salmonella typhimurium.

When a bacterial cell is infected with a phage, the lytic cycle may occur. However, during lytic infection, the enzymes responsible for packaging viral DNA into the bacteriophage sometimes package host DNA accidentally. The result is called a transducing particle. These cannot lead to a viral infection because they contain no viral DNA, and are said to be defective. On lysis of the cell, the transducing particles are released along with normal phages that contain the virus genome. Consequently, the lysate contains a mixture of normal virions and transducing particles. When this lysate is used to infect a population of recipient cells, most of the cells are infected with normal virus. However, a small proportion of the population receives transducing particles that inject the DNA they packaged from the previous host bacterium. Although this DNA cannot replicate, it can recombine with the DNA of the new host. Typically, only about 1 cell in 10⁶ to 10⁸ is transduced for a given marker.

Specialized transduction

On the other hand, some temperate phages can able to take up a specific portion of donor DNA as their part and transduction takes place, which is referred as specialized transduction. The temperate phage (Lambda phage) send its DNA after adsorption. The penetrated viral DNA will get integrated into bacterial chromosomal DNA and persist for ever. This stage of phage is referred as prophage. The cycle is lysogeneic cycle. Due to chemical or physical induction, the prophage get activated, replicated its viral DNA and protein coat followed by assembly and lysis of the cell. The lambda phage will get integrated into bacterial chromosome at specific region (near to gal gene - galactose operon). When induce the prophage, sometimes, the viral DNA wrongly included some portion of bacterial DNA (mostly gal gene) with loss of portion of viral DNA. After assembly, these phages will escape to environment. These phages are designated as lambda (dgal) phages. These l(dgal) phages cannot act as that of normal lambda phages. For infection of recipient cells, the l(dgal) phages need the help of normal lambda phages (referred as helper phages). When these l(dgal) phages infect the E. coli (gal defective gal^-), the transduction take place lead to gain of gal gene. The recipient cells become gal⁺.

Recall how the insertion and excision of prophage took in bacterial chromosome. Improper excision leads to specialized transduction.

Youtube video about generalized transduction

Video describing Specialized transduction (Youtube)

C. Conjugation

Bacterial conjugation (mating) is a process of genetic recombination that involves cell to cell contact. The gene for conjugation (ability to mate) is a plasmid borne character. The gene involves the conjugation is designated as F factor (fertility factor) which located in the plasmid (referred as F plasmid). The cell which has the F plasmid is donor (male), designated as F⁺ cells and cell doesn’t have the F plasmid is the recipient (female), designated as F^-cells. The DNA will be transferred from donor to recipient through physical contact. So another requirement for cell to cell contact is sex pilus through which the donor DNA will be transferred to recipient cell.

The diagrammatic representation shows how the conjugation takes place. The sex pili makes a connection between donor and recipient. A nick (split) in the complementary strand of F plasmid takes place. The single strand plasmid transferred to F^- cell and become double stranded. The F⁺ cell also synthesized its cDNA and become double strand. After conjugation, both the cells become F⁺cells.

Hfr cells

Sometimes, F plasmid transferred from F⁺cell to F^- cell get integrated into recipient cell chromosomal DNA. Such conjugants pocess the F factor in the chromosomal DNA instead of plasmids. They are referred as hfr cells (High Frequency Recombination). They have very high frequency of conjugations.

F’ (F prime) cells

Sometimes, the F plasmid integrated with donor chromosome may excised from the genome accidentally. During this excision process, some portions of chromosomal DNA also incorporated with the original genes of F plasmid, it is called as F’ (variant) plasmid. When, F’ plasmids promote conjugation, they transfer the donor’s chromosomal genes at high frequency to the recipients.

Animation of conjugation (Youtube)

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