Stability of transgenes and presence of N⁶ methyladenine DNA in transformed wheat cells

Abstract

Summary A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post‐selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation‐sensitive enzymes revealed that the DNA represented by the common bands was N6‐adenine methylated (m6A DNA), and there was even m6A DNA in some 30 month samples. Although the source of m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma‐like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.

Adenine methylation atdam sites increases transient gene expression in plant cells

“…Unequivocal demonstration of the existence of 6-methyladenine in eukaryotic nuclear DNAs, based on bulk determinations of modified bases, is lacking. If 6-methyladenine is present the levels are probably low (see Torres et al, 1992 andChen et al, 1994 for discussions). However indirect evidence, based on differential restriction enzyme sensitivities, suggest that 6-methyladenine is presentin eukaryotic genomic DNAs.…”

High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco.

“…This cell line also had high GUS expression (Tables 1 and 2), indicating that in this instance, the rearrangement did not affect gene function dramatically. Chen et al (1994) reported that transgenes in wheat cell lines subjected to direct DNA transfer may sometimes contain A/ 6 -methyladenine. This observation raises the possibility that transformation of an endophyte, such as a mycoplasma-like organism, occurred simultaneously with transformation of the wheat cells.…”

“…That the SAR effect is not transferred from the GUS plasmid to the NPTII plasmid suggests that SARs are somehow functionally separated from the NPTll gene, but we can only speculate about actual integration patterns. Previous reports have shown that cotransformed plasmids frequently integrate near enough to one another to show tight genetic linkage (Chee and Slightom, 1992;Chen et al, 1994;Register et al, 1994;Peng et al, 1995), but few such events have been characterized on a molecular scale. It may be that asmall amount of intervening DNA, such as might be provided by plasmid vector sequences, is all that is necessary to prevent the SAR effect from acting on an adjacent plasmid.…”

pBECKS2000: a novel plasmid series for the facile creation of complex binary vectors, which incorporates “clean-gene” facilities

“…1a), presents a potential target for rearrangements in bacteria. Such an arrangement of a 35S repeat has been noted, during previous studies, to undergo small deletion rearrangements within Agrobacterium but the promoters remained transcriptionally active within transformed plant tissues (Chen et al 1994). Estimation of the sizes of restriction fragments of pBECKS 2000 .3, as mapped in Fig.…”