Recombination: New arrangements of genes
Recombination doesn’t take place only in the laboratory. It is a natural process by which DNA segments are exchanged and rearranged. Recombination is one of the causes of genetic variability of organisms. Recombination occurs particularly frequently among bacteria. Recombination tools are used to remove undesirable marker genes from plants. The first plants on which this kind of system has been used are already in the European authorisation system.
There are several recombination approaches and mechanisms – one of them is homologous recombination. The prerequisite for this are DNA sections with the same or very similar sequence of DNA components. These homologous DNA sequences can be exchanged between two strands. It is also possible for other DNA sections between such homologous sequences to be cut out of one strand and introduced into the other (see diagram).
Unlike with animals and bacteria, homologous recombination occurs only infrequently in plants. Attempts are therefore being made to transfer the bacterial recombination system to plants, where it can be used both to remove marker genes and for the targeted insertion of DNA sequences into the genome.
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The bacterial recombination systems consist of two components:
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Recombinase, an enzyme: since this is not normally present in plants, the gene is introduced into the plants.
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Certain recognition sequences, known as sites: the recombinase recognises “its” sites and causes recombination to occur. A DNA sequence located between the sites is cut out from the DNA strand and can be reinserted at another point – again between two sites.
In order to use recombination systems to eliminate marker genes in transgenic plants, several steps are necessary:
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First, transgenic plants are produced with a marker gene flanked by the sites. If the marker gene is to be removed again, the recombinase gene is also introduced into the plants.
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In progeny that contain both the marker gene and the recombinase gene, the marker gene is removed.
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The recombinase gene can be removed again in the following generation, e.g. by means of segregation.
Several recombination systems are now known. Two of them have been investigated in biosafety research projects to see whether they are suitable for removing marker genes in practice. These results are now available. Another recombination system is the subject of a current research project, which began in 2005.
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The Cre-lox system originates in the P1 bacteriophage. It consists of the Cre recombinase and the lox sites. Depending on the orientation of the lox sites, the gene between them is either cut out or rotated (inversion).
This system has been used with tobacco, sugar beet and potatoes. With potatoes in particular, it was possible to produce marker-free transgenic plants. Since the results were promising, the research is continuing.
GM maize plants in which the marker gene has been removed with the help of the Cre-lox system are now in the European authorisation system. -
The resolvase/res system, another bacterial system, works in a similar way. It cuts the genes out, but without rotating them. The cutting enzyme in this case is a resolvase. The corresponding marker sites are res sequences (see diagram).
A completed SiFo project was able to demonstrate the functionality of the resolvase/res system in potato protoplasts. The system was also optimised for the specific conditions in potato plants.
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