Types of Recombination

I. Naturally occuring recombination

1.) Integration of bacterial DNA fragments - Bacteria have no sexual reproduction in the true sense, but many or most of them are capable of transferring fragments of DNA from cell to cell by one of three machanisms. (1) Fragments of the bacterial genome can become joined to plasmid DNA and transferred by cell conjugation by the same mechanism that secures the transfer of the DNA of transmissible plasmids. (2) Genomic fragments can be carried from cell to cell in the infective coats of bacterial viruses (phages), a process called transduction. (3) Many bacteria have the capicity to assimilate fragments of DNA from solution and so may aquire genes from disrupted cells.

Fragments of DNA acquired by any of these methods can be integrated into the DNA of the genome in place of homologous sequences previously present. If the incoming DNA has no homology with that of the recipient cell, it usually cannot be integrated and is lost for lack of ability to replicate autonomously. Homologous integration in bacteria is similar, in its nonreciprocal nature and perhaps also in its mechanism, to gene conversion in eukaryotic organisms.

2.) Site-Specific Recombination - Bacteriophages, plasmids, bacteria, and unicellular eukaryotes provide many examples of differentiation through controlled and site-specific recombination of DNA segments. Invertebrates, a controlled series of deletions leads to the generation of the great diversity of gene sequences encoding the antibodies and T-cell receptors necessary for immune defense against pathogens. All these processes depend upon interaction and recombination between specific DNA sequences, generally but not always with some sequence similarity, catalyzed by site-specific recombinase enzymes. The molecular mechanisms may have some similarities with those responsible for general meiotic recombination, except that the latter does not depend on any specific sequence, only on similarity (homology) of the sequence recombined.

3.) Nonhomologous Recombination - Techniques have been devised for the artificial transfer of DNA fragments from any source into cells of many different species, thus conferring new properties upon them. In bacteria and the yeast (S. cerevisiae), integration of such DNA into the genome (on which the stability of transformation generally depends) requires substantial sequence similarity between incoming DNA and the recipient site. However, cells of other fungi, higher plants, and animals are able to integrate foreign DNA into their chromosomes with little or no sequence similarity. These organisms appear to have some unidentified system that recombines the free ends of DNA fragments into chromosomes regardless of their sequences. It may have something in common with the mechanism, equally obscure, whereby broken ends of chromosomes can heal by nonspecific mutual joining.

4.) Mitotic Recombination - Crossing-over between homologous chromosome pairs can also occur during the prophase of mitotic nuclear division. The frequency is very much lower than in meiosis, presumably because the mitotic cell does not form the synaptic apparatus for efficient pairing of homologs. Mitotic crossing-over has been studied in the fruit fly Drosophila melanogaster, in the filamentous fungus Aspergillus nidulans, and in Saccharomyces yeast. In these species it is detected through the formation of homozygous clones of cells in an initally heterozygous diploid. There is a 50% chance of homozygosity in daughter cells whenever a cross-over occurs between chromatids in the interval between the marker and the centromere, in the chromosomal site attachment to the mitotic spindle. The frequency of mitotic crossing-over is greatly increased by radiation.

II. Man-made recombination techniques

1.) Plasmid Insertion Recombination - The means by which a plasmid is used to import recombinant DNA into a host cell for cloning. Both a plasmid carrying genes for antibiotic resistance and a DNA strand which contains the gene of interest are cut with the same restriction endonuclease. The plasmid is opened up and the gene is freed from its parent DNA strand. They have complementary "sticky ends." The open plasmid and the freed gene are then mixed with DNA ligase, which reforms the two pieces as recombinant DNA in a variety of configurations, only one of which is desirable. This recombinant DNA stew is allowed to transform a culture of bacteria which is then exposed to antibiotics. All the cells except those which have been encoded by the plasmid DNA are killed, leaving a culture of cells containing the desired recombinant DNA.

2.) Gene Gun Recombination - The gene gun provides an excellent technique for the genetic engineering of plants. It operates on the principle that if DNA molecules coated onto gold particles are bombarded into living cells, this DNA solution can be incorporated into the cell's DNA. A gene gun delivers this DNA solution as a jet stream with sufficient force to penetrate the genome of the target organism.

3.) Virus Replication Recombination - Viruses replicate by invading a host cell and comandeering the unsuspecting cell into producing hundreds of copies of the viral DNA. The virus attaches itself to the outside of the host cell and injects its DNA into the cell. The viral genes are transcribed and translated by the cell's enzymes and ribosomes. The cell does not distinguish between the DNA contributed by the virus and its own DNA; it just follows the genetic instructions available inside of its walls. In this way, the virus takes over the cell's productivity. Now, instead of producing new cell material, the cell has become a virus-producing factory. The new virus particles are eventually released from the cell and go off to find a host cell of their own to take over.

4.) Electric Pulse Recombination (Electroporation) - Foreign DNA has been introduced into plants cells by a novel technique called Electroporation. This technique involves the use of electric pulses to make plant plasma membranes permeable to plasmid DNA molecules. Plasmid DNA taken up this way has been shown to be stably inherited and expressed. This technique has also been used for recombination of animal cells. Carefully controlled electric field pulses are introduced to animal cells. This causes pores to open in the cell membrane allowing genes to gain access to the cell's interior.

12/06/1997