Transgene organization in rice engineered through direct DNA transfer supports a two-phase integration mechanism mediated by the establishment of integration hot spots

Organization of transgenes in rice transformed through direct DNA transfer strongly suggests a two-phase integration mechanism. In the “preintegration” phase, transforming plasmid molecules (either intact or partial) are spliced together. This gives rise to rearranged transgenic sequences, which upon integration do not contain any interspersed plant genomic sequences. Subsequently, integration of transgenic DNA into the host genome is initiated. Our experiments suggest that the original site of integration acts as a hot spot, facilitating subsequent integration of successive transgenic molecules at the same locus. The resulting transgenic locus may have plant DNA separating the transgenic sequences. Our data indicate that transformation through direct DNA transfer, specifically particle bombardment, generally results in a single transgenic locus as a result of this two-phase integration mechanism. Transgenic plants generated through such processes may, therefore, be more amenable to breeding programs as the single transgenic locus will be easier to characterize genetically. Results from direct DNA transfer experiments suggest that in the absence of protein factors involved in exogenous DNA transfer through Agrobacterium, the qualitative and/or quantitative efficiency of transformation events is not compromised. Our results cast doubt on the role of Agrobacterium vir genes in the integration process.

An experimental investigation of liquid distribution and surface character in horizontal annular two-phase flow. OOR Project no. 2117. Contract DA-30-115-ORD-992. Interim report no. 1

Mode of access: Internet.

Proceedings of the second Joint USAEC-EURATOM Two-Phase Flow Meeting. Held at USAEC, Germantown, Maryland, April 29, 30, and May 1, 1964.

At head of title: United States Atomic Energy Commission - USAEC. European Atomic Energy Community - EURATOM.

Evaluation and performance of once-through, zero-gravity boiler tubes with two-phase water /

"Contract AT(30-1)-2789."

Two-phase anaerobic digestion for enhanced stabilization and methanation of sewage sludge /

"ILENR/AE-92/02."

Affinity partitioning of plasmid DNA with a zinc finger protein

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger–GST (Glutathione-S-Transferase) fusion protein was examined in PEG–dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600–DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger–GST fusion protein in a PEG 1000–DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.