Developmentally and transgene regulated nuclear processing of primary transcripts of chalcone synthase A in petunia.

The introduction of chalcone synthase A transgenes into petunia plants can result in degradation of chalcone synthase A RNAs and loss of chalcone synthase, a process called cosuppression or post-transcriptional gene silencing. Here we show that the RNA degradation is associated with changes in premRNA processing, i.e. loss of tissue specificity in transcript cleavage patterns, accumulation of unspliced molecules, and use of template-specific secondary poly(A) sites. These changes can also be observed at a lower level in leaves but not flowers of nontransgenic petunias. Based on this, a model is presented of how transgenes may disturb the carefully evolved, developmentally controlled post-transcriptional regulation of chalcone synthase gene expression by influencing the survival rate of the endogenous and their own mRNA.

Transgene-promoted epigenetic switches of chalcone synthase activity in petunia plants.

Epigenetic variation affecting pigment pattern formation in petunia flowers due to the insertion of transgenes encoding chalcone synthase is described. The loss of pigment formation in petals or parts of petals is due to the post-transcriptional degradation of chalcone synthase RNA, from both the endogenous petunia chalcone synthase genes and from the chalcone synthase transgenes. The RNA cleavage pathway and its control are described. Different epigenetic states of RNA breakdown are correlated with specific cytosine methylation changes in the coding sequences of the genes. The probability, extent and developmental location of chalcone synthase RNA breakdown are related to the number and organization of transgenes in the genome but epigenetic switches that affect RNA turnover probably occur in meristems and between sexual generations. Hypotheses to explain how the transgenes influence the levels of chalcone synthase RNA breakdown and how different epigenetic states are created are discussed.

Cytosine methylation and nucleolar dominance in cereal hybrids.

In wheat-rye hybrids the nucleolus organizer regions (NORs), the sites of ribosomal RNA genes, from rye are suppressed. Wheat and wheat-rye hybrid genetic stocks containing different numbers of wheat and rye nucleolus organizers, as well as addition lines and rye-barley hybrids, were used in Southern hybridization experiments to determine the cause of nucleolar dominance and suppression in cereal hybrids. Based on the use of restriction endonucleases that cleave near the ends of the spacer unit and an additional, methylation-sensitive enzyme, HpaII, which does not recognize the CCGG restriction site if the internal C is methylated, an indirect method of assaying NOR expression was established. The results indicated that cleavage by the HpaII enzyme of the rye NOR sequences, is reduced when major NORs from other cereals were present. The reduction in the number of rye rRNA genes containing an unmethylated CCGG site in the promoter was associated with the suppression of the rye nucleolus. These results are consistent with a model in which promoter and upstream regulatory repeats of ribosomal RNA genes compete for limited concentrations of regulatory proteins, and genes that are methylated at key binding sites fail to engage these regulatory proteins and thus remain inactive.

RNA-mediated RNA degradation and chalcone synthase A silencing in petunia.

Transgenic Petunia plants with a chsA coding sequence under the control of a 35S promoter sometimes lose endogene and transgene chalcone synthase activity and purple flower pigment through posttranscriptional chsA RNA degradation. In these plants, shorter poly(A)+ and poly(A)- chsA RNAs are found, and a 3' end-specific RNA fragment from the endogene is more resistant to degradation. The termini of this RNA fragment are located in a region of complementarity between the chsA 3' coding region and its 3' untranslated region. Equivalent chsA RNA fragments remain in the white flower tissue of a nontransgenic Petunia variety. We present a model involving cycles of RNA-RNA pairing between complementary sequences followed by endonucleolytic RNA cleavages to describe how RNA degradation is likely to be promoted.

Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression.

Analysis of Agrobacterium-transferred DNA (T-DNA) revealed strong correlations between transgene structures and floral pigmentation patterns from chalcone synthase (chs) co-suppression among 47 Petunia transformants. Presented here are the full details of T-DNA structural organization in that population. Sixteen transformants (34%) carried one T-DNA copy while 31 (66%) carried 106 complete and partial T-DNA elements in 54 linkage groups. Thirty linkage groups contained multiple T-DNA copies; 15 of these contained only contiguously repeated copies, 8 contained only dispersed copies and 7 contained both. Right-border inverted repeats were three times more frequent than left-border inverted or direct repeats. Large fragments of binary-vector sequences were linked to the T-DNA in seven plants.

Molecular cloning and characterization of an unusually large intergenic spacer from the Nor-B2 locus of hexaploid wheat.

An allelic rDNA variant from the Nor-B2 locus of 'Bezostaya' wheat that forms an especially active nucleolus was cloned and characterized. It carries an unusually large intergenic spacer compared with rDNA units in most other wheat genotypes. The additional intergenic length is in the array of 135-bp A repeats and not in other internal repeats. These A repeats have sequences nearly identical to other A repeats described for other alleles. It is suggested therefore that the more active Nor-B2 locus of 'Bezostaya' may be due to the constituent rDNA units possessing a larger array of A repeats.

Organization of retro-element and stem-loop repeat families in the genomes and nuclei of cereals.

Sequences homologous to the retro-element BIS-1 and the stem-loop repeat Hi-10 are present in the genomes of a number of cereal species. A detailed characterization of these elements indicated that they are non-randomly organized in the genomes of at least two of these species, namely barley and rye. In contrast to the BIS-1 retro-elements, the stem-loop repeats are also non-randomly organized into discrete domains in interphase nuclei from barley and rye. Features of the organization of these repeats along chromosomes and within interphase nuclei of rye, barley and rice are discussed.

WIS 2-1A: an ancient retrotransposon in the Triticeae tribe.

WIS 2-1A, the first retrotransposon found in wheat, has been recently studied and characterized. Southern hybridization experiments utilizing several species from the Triticeae revealed the presence of homologous sequences in all the taxa tested, showing high levels of interspecific variability and almost no intraspecific differentiation. Further experiments using in situ hybridization in several species showed that the retroposon was almost completely dispersed throughout the genomes tested. These results suggested that WIS 2-1A is an ancient element that probably was present in the unknown common ancestor of the Triticeae and that only under rare circumstances does it become active. DNA fragments homologous to the WIS 2-1A reverse transcriptase gene were isolated from most of the Triticeae species using PCR. The fragments obtained were sequenced and analyzed. Even though the sequence alignment was consistent with the phylogenetic studies made in the past, the genus Thinopyrum showed new evidence for a possible horizontal propagation of the retroelement.Mention of a trade name or proprietary product does not constitute a guarantee, warranty or recommendation of the product by the U.S. Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitableAll programs and services of the U.S. Department of Agriculture are offered on a non-discriminatory basis without regard to race, colour, national origin, religion, sex, age, marital status, or handicap.

Inactivation of gene expression in plants as a consequence of specific sequence duplication.

Numerous examples now exist in plants where the insertion of multiple copies of a transgene leads to loss of expression of some or all copies of the transgene. Where the transgene contains sequences homologous to an endogenous gene, expression of both transgene and endogenous gene is sometimes found to be impaired. Several examples of these phenomena displaying different features are reviewed. Possible explanations for the observed phenomena are outlined, drawing on known cellular processes in Drosophila, fungi, and mammals as well as plants. It is hypothesized that duplicated sequences can, under certain circumstances, become involved in cycles of hybrid chromatin formation or other processes that generate the potential for modification of inherited chromatin structure and cytosine methylation patterns. These epigenetic changes could lead to altered transcription rates or altered efficiencies of mRNA maturation and export from the nucleus. Where the loss of gene expression is posttranscriptional, antisense RNA could be formed on accumulated, inefficiently processed RNAs by an RNA-dependent RNA polymerase or from a chromosomal promoter and cause the observed loss of homologous mRNAs and possibly the modification of homologous genes. It is suggested that the mechanisms evolved to help silence the many copies of transposable elements in plants. Multicopy genes that are part of the normal gene catalog of a plant species must have evolved to avoid these silencing mechanisms or their consequences.

Partial characterization of the Nicotiana tabacum actin gene family: evidence for pollen-specific expression of one of the gene family members.

The actin gene family of Nicotiana tabacum has been partially characterised by Southern hybridisation and by isolating lambda EMBL4 recombinants from a genomic library having homology to the soybean actin gene, Sac3. The number of actin genes with homology to Sac3 is estimated at between 20 to 30, based on Southern hybridisation and library screening, though the total gene family may be larger. Twenty-four recombinant lambda clones were isolated, 18 had unique restriction profiles and from these, 2 clones, Tac9 and Tac25, were selected for further study. The region of Tac25 hybridizing to Sac3 was sequenced and shown to contain an open reading frame (ORF) with homology to actin. Partial sequencing of Tac9 revealed a sequence with homology to the third exon of Tac25 and Sac3. The two tobacco actin sequences were compared to other reported actin gene sequences; Tac25 was closely related to the allelic potato actins, Pac58 and Pac85, while Tac9 was more related to Pac79 than to other plant actins. Northern hybridisation analysis showed that while Tac9 detected actin transcripts in RNA from root, leaf, stigma and pollen, Tac25 transcripts were only detected in pollen RNA.

Sequence analysis of WIS-2-1A, a retrotransposon-like element from wheat.

WIS-2-1A, a 8624 bp insertion in the Glu-1A-2 locus of chromosome 1A of wheat, consists of two 1755 bp long terminal repeats enclosing a 5114 bp internal region. No long open reading frames could be found, but inspection of the predicted amino acid sequence showed regions with homology to retrotransposon structures, including a methionine tRNA initiator binding site, a nucleotide binding domain, a protease, an integrase and a polymerase. DNA replication errors have resulted in frame-shifts in the protein coding region, suggesting that retrotransposition of WIS-2-1A, if it occurs, must be mediated by trans-acting factors.

Protein-binding to reiterated motifs within the wheat rRNA gene promoter and upstream repeats.

The binding of specific proteins in wheat nuclear extracts to the -34 to +10 region of the wheat ribosomal RNA gene promoter has been detected. DNase I footprinting revealed two binding regions, one spanning the transcription initiation site from -7 to +2, and a larger one further upstream from around -29 to -15. An oligonucleotide made to the -30 to -14 promoter region binds a protein in a sequence-specific manner. This protein also binds specifically to sites with related sequences further upstream in the promoter and in the associated intergenic spacer repeats. A consensus sequence, CATGG--GC-AAAAC, was defined from the different binding sites of the protein. These results, taken together with in vivo results on the enhanced activity of nucleoli containing ribosomal RNA genes with more intergenic spacer repeats, endorse the hypothesis that ribosomal RNA gene transcription is regulated by the extent of binding of a protein to many sites upstream of the transcriptional start site.

Inverted repeats in the long-terminal repeats of the wheat retrotransposon Wis 2-1A.

The wheat insertion sequence Wis 2-1A possesses all the structural features characteristic of retrotransposons. Its long-terminal repeats (LTRs) are unusually long (1,755 bp) compared with those of other retrotransposons. Sequence analysis revealed that they differ from each other by only six point mutations. They contain a few tandem direct repeats, which could be explained by slippage mechanisms during replication. Almost half (44%) of the length of the LTRs is occupied by hairpin structures, which may relate to their large size. Possible origins of these inverted repeats are proposed, including the insertion and imprecise excision of transposable elements and errors when the DNA replication intermediate switches RNA template during retrotransposon replication.

Analysis of HMW glutenin subunits encoded by chromosome 1A of bread wheat (Triticum aestivum L.) indicates quantitative effects on grain quality.

A gene encoding the high-molecular-weight (HMW) subunit of glutenin 1Ax1 was isolated from bread wheat cv Hope. Comparison of the deduced amino acid sequence with that previously reported for an allelic subunit, 1Ax2(*), showed only minor differences, which were consistent with both subunits being associated with good bread-making quality. Quantitative analyses of total protein extracts from 22 cultivars of bread wheat showed that the presence of either subunit 1Ax1 or 1Ax2(*), when compared with a null allele, resulted in an increase in the proportion of HMW subunit protein from ca. 8 to 10% of the total. It is suggested that this quantitative increase in HMW subunit protein may account for the association of 1Ax subunits with good quality.

Identification of an enhancer element for the endosperm-specific expression of high molecular weight glutenin.

Genes encoding high molecular weight (HMW) glutenin, a wheat seed storage protein, are expressed only in the developing endosperm. It was previously demonstrated that sequences essential for endosperm-specific transcription reside within 436 base pairs upstream of the initiation codon for HMW glutenin translation. We have further analyzed this region by testing the ability of a series of truncated HMW glutenin promoter fragments to enhance transcription from an adjacent heterologous promoter. The activity of these hybrid promoters was determined by measuring the expression of a linked beta-glucuronidase (GUS) reporter gene in transgenic tobacco plants. An HMW glutenin promoter fragment spanning nucleotides -375 to -45 relative to the transcription start site was found to stimulate GUS expression in tobacco seeds when inserted in either orientation upstream of the heterologous promoter. Furthermore, this fragment could also potentiate transcription when located 3' to the GUS reporter gene. Stimulation of GUS gene expression in transgenic tobacco seeds did not occur until 9 days to 12 days after anthesis, coincident with the onset of storage protein synthesis in the developing tobacco and wheat seed, and was confined to the endosperm tissue. By testing progressively shorter promoter fragments, the enhancer element responsible for this pattern of expression was localized to a 40-base pair region some 170 base pairs upstream of the start site for HMW glutenin transcription.