Condensation of chromatin in transcriptional regions of an inactivated plant transgene: evidence for an active role of transcription in gene silencing.

The chromatin structures of two epigenetic alleles of a transgene were investigated by measuring the local accessibility of transgene chromatin to endonucleases. The two epialleles represented the active, hypomethylated state of a transgene in line 17-I of Petunia hybrida, and a transcriptionally inactive, hypermethylated derivative of the same transgene in line 17-IV. In nuclear preparations the inactive epiallele was significantly less sensitive to DNasel digestion and nuclease S7 digestion than the transcriptionally active epiallele, whereas no significant differences in accessibility were observed between naked DNA samples of the two epialleles. Our data suggest that a condensed chromatin structure is specifically imposed on transcribed regions of the construct in line 17-IV. In contrast, in both epialleles the plasmid region of the transgene, which is not transcriptionally active in plants, retains the same accessibility to endonucleases as the chromosomal integration site. These data suggest that transcriptional inactivation is linked to the process of transcription, and imply that control of transgene expression via the use of inducible or tissue-specific promoters might prevent transgene silencing and conserve the active state of transgenes during sexual propagation.

A repetitive DNA fragment carrying a hot spot for de novo DNA methylation enhances expression variegation in tobacco and petunia.

A 1.6 kb repetitive DNA sequence (RPS) from Petunia hybrida was identified that destabilizes expression of a GUS marker transgene. Following polyethylene glycol (PEG)-mediated tobacco and petunia protoplast transformations, GUS expression patterns analysed on callus and plant levels were clearly more variable when constructs contained the RPS sequence. The effect on transgene expression required chromosomal integration since the two different RPS constructs employed did not exhibit reduced levels of GUS activities in transient assays. DNA methylation analysis implies a hypermethylated default state of endogenous RPS copies present in the petunia genome. Analysis of the transgene DNA in different transgenic tobacco plants showed almost complete hypermethylation of a particular Hhal site of the RPS sequence. It is proposed that, due to the presence of specific signals within the RPS region or based on interaction of RPS with other endogenous homologous sequences, RPS functions as an initiation region for de novo methylation and induces expression variegation in adjacent sequences.

Systematic relationships within the Anthozoa (Cnidaria: Anthozoa) using the 5'-end of the 28S rDNA.

Systematic relationships among the subclasses of Anthozoa, and especially among the orders Scleractinia, Actiniaria, and Corallimorpharia of subclass Zoantharia, were investigated by applying parsimony and distance methods of analysis to nucleotide sequence data obtained for the 5' end of the 28S rDNA. Exhaustive parsimony analysis indicates that the Ceriantipatharia are most representative of the ancestral Anthozoa. When applied to a wide range of scleractinians (nine taxa), actiniarians (seven taxa), and corallimorpharians (six taxa), both parsimony and distance analyses resolve three groups, one being the Scleractinia and the others containing both actiniarians and corallimorpharians. This indicates an unclear relationship between Actiniaria and Corallimorpharia and gives no support for Hand's hypothesis of scleractinian ancestry of actiniarians and corallimorphians; the monophyly of the Scleractinia, which is strongly supported by our analyses, is evidence to the contrary.

Treatment with propionic and butyric acid enhances expression variegation and promoter methylation in plant transgenes.

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.

Evidence for cytosine methylation of non-symmetrical sequences in transgenic Petunia hybrida.

A considerable proportion of cytosine residues in plants are methylated at carbon 5. According to a well-accepted rule, cytosine methylation is confined to symmetrical sequences such as CpG and CpNpG, which provide the signal for faithful transmission of symmetrical methylation patterns by maintenance methylase. Using a genomic sequencing technique, we have analysed cytosine methylation patterns within a hypermethylated and a hypomethylated state of a transgene in Petunia hybrida. Examination of a part of the transgene promoter revealed that in both states m5C residues located within non-symmetrical sequences could be detected. Non-symmetrical C residues in the two states were methylated at frequencies of 5.9 and 31.9%, respectively. Methylation appeared to be distributed heterogeneously, but some DNA regions were more intensively methylated than others. Our results show that at least in a transgene, a heterogeneous methylation pattern, which does not depend on symmetry of target sequences, can be established and conserved.

Nucleotide sequence of the histone gene cluster in the coral Acropora formosa (Cnidaria; Scleractinia): features of histone gene structure and organization are common to diploblastic and triploblastic metazoans.

We report the nucleotide sequence of the core histone gene cluster from the Cnidarian Acropora formosa. This is the first histone gene cluster to be sequenced from a diploblastic organism and the predicted amino acid sequences most resemble those of sea urchin equivalents. Each of the Cnidarian histone genes has two conserved regions 3' of the coding sequences and these closely resemble those of the metazoan alpha-class histone genes. In A. formosa the core histone genes are arranged as opposed (H3/H4 and H2A/H2B) pairs, a pattern common to the nondeuterostome metazoa, and tandem repetition is the predominant pattern of organization in the Cnidarian. With the recent identification of several classes of homeobox genes in Cnidarians these features clearly align the Cnidaria with triploblastic metazoans, supporting a monophyletic origin of the metazoa.

Nucleotide sequence of the histone H3-encoding gene from the scleractinian coral Acropora formosa (Cnidaria: Scleractinia).

The histone H3-encoding gene from the staghorn coral Acropora formosa has the 3'-hairpin structure which functions as termination/processing signal in nonpolyadenylated histone mRNAs. The predicted amino acid sequence is highly homologous with the major H3 histones of higher metazoans, and has least homology with the corresponding protist and fungal proteins. These data are inconsistent with the phylogenetic position which has been assigned to the Cnidaria by partial 18S rRNA sequence comparison.