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.

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.

DNA fingerprinting in sugar beet (Beta vulgaris) - identification of double-haploid breeding lines.

The distribution and abundance of simple repetitive sequences complementary to the synthetic oligonucleotides (GACA)4, (GATA)4, (GTG)5 and (CA)8 in the genomes of several cultivars of Beta vulgaris and in the wild beet B. vulgaris ssp. maritima were investigated. Hybridization experiments revealed that all four motifs were present, though at different abundances, in the genomes of all of the investigated beet cultivars. Considerable intraspecific variation of the resulting DNA fingerprints was observed. The extent of polymorphism depends on the oligonucleotide probe. The most informative banding patterns were obtained with the (GATA)4 probe hybridized to HinfI-, HaeIII-, or RsaI-restricted DNA, respectively. DNA fingerprinting with (GATA)4 allowed a clear differentiation of double-haploid breeding lines (DH lines). We demonstrated that the application of oligonucleotide probes for DNA fingerprinting is a sensitive tool for genome diagnosis in cultivated beet.

RNA editing in tobacco chloroplasts leads to the formation of a translatable psbL mRNA by a C to U substitution within the initiation codon.

The psbL gene which codes for a 38 amino acid peptide of photosystem II, together with the photosynthetic genes psbE and psbF, is contained in a conserved position of many species of higher plant plastomes. The alignment of the psbL nucleotide sequences from ten species shows strong conservation, which is indicative of a functional gene. The tobacco and spinach psbL genes have, however, an ACG codon instead of the initiator ATG codon observed in the homologous position of the other eight species. Evidence is presented that in tobacco chloroplasts a translatable psbL mRNA containing an AUG initiator codon is formed by a C to U editing of the ACG codon. This observation, following the previously reported editing of an rpl2 gene in maize chloroplasts, underlines a more widespread occurrence of this type of posttranscriptional mRNA modification and demonstrates its presence in a dicotyledon plant.

Repeated DNA sequences of Aegilops markgrafii (Greuter) Hammer var. markgrafii: cloning, sequencing and analysis of distribution in Poaceae species.

Total DNA of Aegilops markgrafii (Greuter) Hammer var. markgrafii was shot gun cloned. From all the recombinants containing repetitive sequences 1-2% hybridized preferentially with the Ae. markgrafii genome and were almost absent in wheat. The cloned sequences are disperse distributed over the Aegilops chromosomes and show the typical features of eukaryotic repetitive DNA. Five specific probes were tested for their applicability in a screening program on 68 Poaceae accessions.

Cloning and characterization of a Beta vulgaris satellite DNA family.

Three members of the BamHI-sequence family of sugar beet (Beta vulgaris) have been cloned in Escherichia coli and compared by sequence analysis. The sequence family shows the typical features of eukaryotic satellite DNA, e.g., organization in tandem arrays and sequence divergence. A typical ladder pattern of a monomer (327 bp) and multiple oligodeoxyribonucleotides have been observed. The BamHI monomer is A + T-rich (69%) and does not show any similarity to other known plant satellite DNAs.

Distribution and evolution of two satellite DNAs in the genus Beta.

EcoRI monomers of a highly repetitive DNA family of Beta vulgaris have been cloned. Sequence analysis revealed that the repeat length varies between 157-160 bp. The percentage of AT-residues is 62% on average. The basic repeat does not show significant homology to the BamHI sequence family of B. vulgaris that was analyzed by us earlier. Both the EcoRI and BamHI sequences are investigated and compared to each other with respect to their genomic organization in the genus Beta. Both repeats were found to be tandemly arranged in the genome of B. vulgaris in a satellite-like manner. The EcoRI satellite DNA is present in three sections (Beta, Corollinae and Nanae) of the genus, whereas the BamHI satellite DNA exists only in the section Beta. The distribution of the EcoRI and BamHI satellite families in the genus is discussed with respect to their evolution.

Construction of Beta procumbens-specific DNA probes and their application for the screening of B. vulgaris x B. procumbens (2n = 19) addition lines.

Beta procumbens-specific DNA probes have been constructed by cloning digested total DNA in E. coli and screening the resulting recombinant plasmids in dot blot hybridizations with labelled B. procumbens and B. vulgaris DNA. Four clones (pTS1-4) have been analyzed in detail determining their degree of specificity and DNA sequence. Two clones (pTS1 and pTS2) with the highest degree of B. procumbens specificity were adapted for the squash dot hybridization with the aim of screening large numbers of individual hybrid plants (B. vulgaris x B. procumbens) carrying an alien B. procumbens chromosome (2n = 19). These addition lines carry in some cases B. procumbens resistance genes to the beet cyst nematode (Heterodea schachtii Schm.).

Genome specific, highly repeated sequences of Hordeum vulgare: cloning, sequencing and squash dot test.

Highly repeated sequences of nuclear DNA from barley Hordeum vulgare (L.) variety 'Erfa' were cloned. Several clones containing barley specific repeated DNA were analysed by sequence analysis and Southern blot hybridization. The investigated repeats differ from each other in their length, sequence and redundancy. Their length ranges from 36 bp to about 180 bp. The repeats are AT-rich and differ widely in their redundancy within the barley genome. Southern analysis showed that the repeats belong to different repetition complexes. The possibility for utilizing these clones as probes for simple and fast genome analysis is demonstrated in squash dot experiments.

Comparative restriction endonuclease analysis and molecular cloning of plastid DNAs from wild species and cultivated varieties of the genus Beta (L.).

A phyletic tree of the genus Beta has been constructed based on EcoRI and PstI plastid DNA restriction patterns of eight species from three sections of the genus. In contrast to the remarkable morphological variability of the varieties of B. vulgaris the restriction patterns of the plastid DNA of this species were found to be almost identical. The comparison of plastic DNAs of B. vulgaris crassa fertile and sterile lines with 13 different restriction enzymes revealed only a single fragment polymorphism in the HindIII patterns. Hybridization analyses in the plastidal rDNA region revealed an interesting loss of an EcoRI restriction site in all cultivated B. vulgaris varieties in contrast to wild species. The results of the construction of clone banks for SalI and BamHI fragments of plastid DNA from fertile B. vulgaris crassa are reported and difficulties in the cloning of specific fragments are discussed.

Wheat specific repetitive DNA sequences - construction and characterization of four different genomic clones.

The construction and molecular analysis of four recombinant clones - pTa1, pTa2, pTa7, and pTa8 - is described. The four clones contain different highly repeated sequences of genomic DNA from Triticum aestivum variety 'Chinese Spring'. The wheat specificity has been determined by colony and dot blot hybridization in comparison with total rye DNA (Secale cereale variety 'Petka'). The four clones with a variable degree of specificity were compared by sequence analysis after the recloning of wheat DNA inserts into M13 mp8. Within the sequencing data a tendency can be observed that those repeated sequences which show the highest degree of species specificity contain a significantly increased amount of GC residues.

A transfer RNAArg gene of Pelargonium chloroplasts, but not a 5S RNA gene, is efficiently transcribed after injection into Xenopus oocyte nuclei.

We present the primary structure of a chloroplast tRNAArgACG gene of the plant, Pelargonium zonale, and its faithful expression in Xenopus oocyte nuclei. This tRNAArg gene is located 250 bp downstream of a 5S RNA gene within a cloned 5kb long ribosomal DNA segment (Fig. 1). The Pelargonium tRNAArg gene shares 97% and 86% sequence homology with tRNAArgACG genes of Spirodela oligorhiza and Euglena gracilis chloroplasts, respectively, and also extensive homology (70%) with the corresponding gene of E. coli. It lacks an intervening sequence and, like eukaryotic tRNA genes, does not code for the 3' terminal CCA nucleotides. Moreover, the chloroplast tRNAArg gene carries all the sequence elements essential for transcription by vertebrate RNA polymerase III since it is efficiently expressed in Xenopus oocyte nuclei, even in the presence of 1 microgram/ml alpha-amanitin. In Xenopus oocyte nuclei, no transcripts of the chloroplast 5S RNA gene were detected.

Variations of chloroplast DNAs in the genus Pelargonium and their biparental inheritance.

The comparison of EcoRI patterns of chloroplast DNAs (ctDNAs) from five species of the genus Pelargonium and from 16 cultivars and varieties of Pelargonium zonale hort. demonstrates a remarkable inter- and intraspecific ctDNA (plastome) variation. The plastome of the P. zonale varieties could be differentiated into groups I, II and III. Reasons for this variation seem to be: occurrence of numerous spontaneous plastome mutations, intense hybridisation by gardeners and breeders, and biparental plastid inheritance.Crosses of P. zonale varieties with different ctDNA types lead to the direct evidence on the molecular level of biparental plastid inheritance and plastid sorting-out in F1-hybrids.