Marc Cornelissen

cry IA(b) transcript formation in tobacco is inefficient.

Chimaeric PCaMV35Scry genes direct in tobacco mesophyll protoplasts mRNA levels of less than one transcript per cell. We provide evidence that this low cytoplasmic cry IA(b) mRNA level is not due to a rapid turnover but rather results from a marginal import flow of cry messenger into the cytoplasm. Run-on assays indicate that the frequency of transcription initiation is not limiting. However, the cry precursor mRNA carries at least three regions that are recognized as introns. The absence of high cytoplasmic levels of spliced cry mRNAs suggests that these mRNAs are unstable and/or not efficiently made. Point mutations in the 5′ splice site of the most distal intron allows high accumulation levels of the full-length mRNA. This implies that the inefficient formation of full-size mRNA is a major cause of the low expression level of chimaeric cry IA(b) genes in tobacco.

Plastid Transformation: A Progress Report

Reproducible methods, allowing the introduction of defined DNA fragments in the plant nuclear genome, are presently available (see previous chapters in this volume) and are contributing significantly to the experimental analysis of the gene structure-function relationships governing regulated gene-expression in plants.

A 6′ gentamicin acetyltransferase gene allows effective selection of tobacco transformants using kanamycin as a substrate

Abstract6′ gentamicin acetyltransferases detoxify aminoglycoside antibiotics containing a 6′ amino group. We tested whether a 6′ gentamicin acetyltransferase gene (6′ gat) of Shigella sp. is suitable as selectable gene in plant transformation using kanamycin (Km) as a substrate. A comparative transformation experiment using Nicotiana tabacum SR1 protoplasts showed that 6′ gat is as effective for selection of transformants as the commonly used neomycin phosphotransferase II (nptII). In stably transformed plants we detected moderate levels of the 6′ gat mRNA. An enzymatic assay was developed with which the acetyltransferase activity of the protein is easily demonstrated.

Co-suppression of β-1,3-glucanase genes in Nicotiana tabacum

Recent studies have demonstrated that introduction of sense gene constructs in plants can result in transgenic plants which express the transgene but also in plants with suppressed levels of transgene expression (reviewed by Jorgensen 1991; Kooter and Mol 1993; Matzke and Matzke 1993). In many silencing cases, the transgene and homologous host genes are coordinately suppressed in the plants, a phenomenon called co-suppression (Napoli et al. 1990). Although the underlying mechanism(s) are unknown, co-suppression has been reported in different plant species, and thus may represent a new approach to the manipulation of gene expression in plants. The finding that the presence of transgenes can influence the expression of resident genes via a homology-based mechanism opens up the possibility that this type of gene regulation may be naturally occurring in plants. A major challenge now is to unravel the molecular mechanism(s) responsible for transgene-induced gene silencing in plants.