Marker gene elimination

Jumping genes: from phenomenon to tool

Some genes can jump. This ability can be exploited to remove undesirable marker genes from genetically modified plants.

For a long time, geneticists believed that genes were fixed at particular sites in a chromosome. In 1949 Barbara McClintock destroyed this belief: she discovered the "jumping genes" – genes that change their position and can even move to other chromosomes. She received the Nobel Prize for this discovery in 1983.

The traces of jumping genes. Maize kernels of different colours

Later, people discovered that such mobile genetic elements occur not only in maize, but also in lots of other organisms. Not only that, but that transposons – the scientific name for jumping genes – play an important role in biology, because they contribute to the genetic variability of organisms.

Later, the processes that enable certain genes to jump were described at the molecular biological level: there are various "transposable elements" which ensure that genes – e.g. those that determine the colour of maize kernels – leave their position in the chromosome and rejoin it in a different location.

The transposons that have been most comprehensively characterised are those of the "Ac/Ds family". The aim is to develop new tools using these transposons, in order to be able to e.g. remove marker genes from transgenic plants after transformation . Ac/Ds transposons occur naturally in maize, but they continue to function even if used in other plant families.

A transposon contains a gene for a special enzyme (Ac transposase), which recognises certain signals (Ds sequences) in the DNA, cuts pieces out of the DNA at these points and reintegrates them in the genetic matter at a different, unpredictable site. The transposase causes a gene to jump only if a Ds signal is present (see diagram below).

To be able to use this Ac/Ds system mechanism for the subsequent elimination of marker genes the following steps must be carried out:

Step 1: Construction of special vector constructs: In order for the Ac transposase enzyme to be able to cut out a particular gene, the gene must be marked with Ds signal sequences. The vector used to transform the plant needs to contain two sections: one with the target gene (GEN), flanked by two Ds sequences, the other with the marker gene (in this case a PAT gene which confers herbicide resistance ) and the AC gene which is responsible for the formation of the transposase enzyme.

If the plant has taken up the vector and integrated the gene construct into its own genome then both sections will be present there. The target and marker genes are next to each other on the plant chromosome, as they were on the vector.

Step 2: Separating the two DNA sections: Now the Ac/Ds system can become active in the plant: the Ac transposase separates the gene construct at the sites with the Ds signals. Then the gene between the Ds sequences is moved to a different part of the genome and integrated there. The integration site is non-specific and cannot be determined in advance. As soon as the marker and target genes are located on different chromosomes they are separate.

If these plants are crossed with other plants, they will produce progeny which, as a result of naturally occurring segregation processes, carry either only the target gene with minimal Ds sequences at both ends, or the marker gene (in this case PAT) with the Ac gene. This achieves the desired result: genetically modified plants into which only the target gene has been inserted.

Results: It has been demonstrated in two research projects that the Ac/Ds transposon system works in principle in sugar beet. However, a few questions still need to be answered before a practical application of the system is possible. These are to be investigated in a follow-up project. A decision still has to be made as to which gene should be made to jump: the target gene or the marker gene.

Nutzung von Transposons beim Gene targeting. Transposons werden nicht nur für die Markergen-Eliminierung eingesetzt, sondern auch beim Gene targeting, dem gezielten Einbau von Genen ins Pflanzengenom. Ein neuer Ansatz beim Gene targeting besteht darin, das Zielgen mit Hilfe eines Transposonsystems in das Pflanzengenom zu integrieren, da Transposons in der Regel in genomische Regionen mit höherer Gen-Expression integriert werden als T‑DNA s.

Jumping genes: from phenomenon to tool

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