Search results

Search:
in the keywords: INTEGRATION

Sort By:

Limit search:

Transgene expression in plant cell

100%

Niemirowicz-Szczytt K., Bartoszewski G.

Biotechnologia

2000

issue 4

11-23

Transgene incorporation in plant genome does not mean that the gene will be active and will show expression at the required level. It is often the case that transgenic plants do not exhibit the activity of the gene introduced. Gene expression is known to be dependent on many factors such as: chimeric gene structure, proper and stable integration to the genome and proper transcription and translation. The knowledge of transgene regulation is essential for the understanding of the transcription and translation mechanisms. Each plazmid component is vital for later transgene expression. This refers to the size of T-DNA introduced, location of particular elements, leader sequences length, AUG sequences, introns presence, optimum coding gene synthesis, removal of RNA instability signals and many other possible modifications.

Agrobacterium T-DNA in the eukaryotic cell: nuclear import and integration into the plant genome

100%

Ziemienowicz A.

Biotechnologia

2000

issue 4

24-31

Agrobacterium tumefaciens, a gram-negative soil bacterium, is able to transfer DNA to most plant species causing crown gall disease in dicotyledonous plants. Due to this activity Agrobacterium is widely used for plant transformation. The transferred DNA (T-DNA) that resides on a large Ti plasmid is processed within the bacterium and is exported to the plant where it is integrated into the chromosome. DNA transfer requires plasmid encoded virulence (vir) genes as well as several chromosomal genes. In vivo studies suggested that Agrobacterium proteins are involved in T-DNA transfer and integration. We study the function of virulence proteins VirD2 and VirE2 in T-DNA nuclear import and integration using in vitro systems. We found that the T-DNA is imported into the plant cell nucleus as a complex with VirD2 and VirE2 proteins. The C-terminal NLS of VirD2 has a piloting function in this process. Import of the T-DNA follows the classical NLS- and importin-dependent nuclear import pathway for proteins. For studies of integration of T-DNA into the plant DNA an in vitro integration/ligation assay has been designed. We have found out that VirD2 is not able to ligate the T-DNA to the plant DNA in vitro. Consequently, plant enzymes must be involved in this process. Indeed, we found an activity responsible for the ligation of T-DNA in extracts from tobacco BY2 suspension cultured cells and from pea axes. This activity is likely to originate from plant DNA ligase, since the T-DNA ligation shows the same requirements for hydrolysis of ATP to AMP as ligation mediated by any ATP-dependent DNA ligase. This does not, however, exclude the involvement of other plant enzymes in T-DNA integration.

Refine search results

2 Biotechnologia

2 2000

1 Bartoszewski G.

1 Niemirowicz-Szczytt K.

1 Ziemienowicz A.

Search results

Transgene expression in plant cell

Agrobacterium T-DNA in the eukaryotic cell: nuclear import and integration into the plant genome