ABSTRACT
The transformation booster sequence (TBS) from Petunia hybrida enhances transformation frequencies in P. hybrida, Nicotiana tabacum and Zea mays. TBS also stimulates homologous inter- and intramolecular recombination in P. hybrida, the molecular basis for this stimulation is not known. We investigated whether TBS contains sequence elements that might contribute to the stimulation of recombination and whether its recombinogenic potential reflects a biological function of TBS. We identified a scaffold attachment region (SAR) within TBS and analysed its distribution in the genome and its homologies to other genomic sequences. A 516 bp subfragment of TBS binds to the nuclear scaffold. The sequence of the TBS-SAR fragment shows strong homologies to retroviral elements from plants, suggesting that TBS is an inactive derivative of a retrovirus that still promotes DNA recombination.
Subject(s)
Plant Proteins/genetics , Plants/genetics , Retroelements/genetics , Base Sequence , Cell Nucleus/genetics , Cell Nucleus/metabolism , Molecular Sequence Data , Plant Proteins/metabolism , Plants/metabolism , Recombination, Genetic/geneticsABSTRACT
Two phenotypic marker genes (A1 and GUS) were employed to monitor the influence of small chain fatty acids on transgene expression in petunia and tobacco. In plants homozygous with respect to the A1 transgene, which normally carry red flowers due to A1 expression, fatty acid treatment induced a range of variegated and white pigmentation patterns which persisted for several months after terminating the treatment. The inhibitory effect was clearly less pronounced for heterozygous plants of the same transgenic line. In all cases the reduction of transgene activities correlated with an increase in transgene promoter methylation. Contrary to evidence reported for mammals and Drosophila, we do not observe an increase in gene expression, but an enhancement of DNA methylation and epigenetic variegation. The inhibition of transgene activity was also observed in several tobacco transformants cultured on fatty acid containing media. Some tobacco transformants carrying Gus-coding regions driven by either 35S or 1'2' promoters responded with a significant reduction in GUS activity. This study suggests that, rather than exerting a general response on all chromatin regions, fatty acids appear to affect genes in a labile epigenetic surrounding or all genes in a susceptible chromatin configuration. Thus, application of these agents may be useful to screen and monitor transgenes prone to epigenetic instabilities.
Subject(s)
Butyrates/pharmacology , Gene Expression Regulation, Plant/drug effects , Genes, Plant/drug effects , Plants/metabolism , Promoter Regions, Genetic/drug effects , Propionates/pharmacology , Transformation, Genetic/drug effects , Acetylation , Blotting, Southern , Butyric Acid , Chromatin/metabolism , DNA, Plant/metabolism , Electrophoresis, Polyacrylamide Gel , Histones/metabolism , Methylation , Pigmentation/genetics , Plants/genetics , Plants, Toxic , Nicotiana/genetics , Nicotiana/metabolismABSTRACT
Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated M(r) of 38,777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.
