Mutants involved in floral and plant development in Petunia

The Petunia system is described together with its possible use for molecular research on floral development. Developmental mutants are described in some detail. The most interesting mutants are Blind and Green petals. In both the appearance of only the petals has changed, in Blind towards antheroid tissue, in Green petals towards sepals. Transposon tagging as an approach to isolate developmental genes in Petunia is also discussed.     

Chromosome landing at the Arabidopsis TORNADO1 locus using an AFLP-based strategy

The Arabidopsis tornado1 (trn1) mutation causes severe dwarfism combined with twisted growth of all organs. We present a chromosome landing strategy, using amplified restriction fragment length polymorphism (AFLP) marker technology, for the isolation of the TRN1 gene. The recessive trn1 mutation was identified in a C24 transgenic line and is located 5?cM from a T-DNA insertion. We mapped the TRN1 locus to the bottom half of chromosome 5 relative to visible and restriction fragment length polymorphism (RFLP) markers. Recombinant classes within a 3-cM region around TRN1 were used to build a high-resolution map in this region, using the AFLP technique. Approximately 300 primer combinations have been used to test about 26?000 fragments for polymorphisms. Seventeen of these AFLP markers were identified in the 3-cM region around TRN1. These markers were mapped within this region using individual recombinants. Four of these AFLP markers co-segregate with TRN1 whereas one maps at one recombinant below TRN1. We isolated and cloned three of these AFLP markers. These markers identified two yeast artificial chromosome (YAC) clones, containing the RFLP marker above and the AFLP marker below TRN1, demonstrating that these YACs span the TRN1 locus and that chromosome landing has been achieved, using an AFLP-based strategy.     

Localization of T-DNA Insertions in Petunia by Fluorescence in Situ Hybridization: Physical Evidence for Suppression of Recombination

Using fluorescence in situ hybridization (FISH) with metaphase preparations, we localized a 4-kb single-copy T-DNA sequence in a group of petunia transformants. The selected T-DNAs previously had been shown to be linked to the phenotypic marker FI on chromosome II. Linkage analysis had revealed that recombination around the FI locus is suppressed in a wide cross relative to an inbred recombination assay. The localization of six FI-linked T-DNAs and the FI locus itself, using FISH, revealed a number of aspects of recombination in petunia: (1) the central region of chromosome II showed at least a 10-fold suppression of recombination in wide crosses relative to the distal region; (2) recombination in wide hybrids over two-thirds of the chromosome was extremely low; and (3) recombination between completely homologous chromosomes in an inbred cross also was suppressed in the central region. In addition, the T-DNAs were not evenly distributed along the chromosome, suggesting a possible preference for a distal position for T-DNA integration. Implications for such a preference are discussed.     

Regulation of gene action in Petunia hybrida: Unstable alleles of a gene for flower colour

Summary In a progeny of a selfed individual of the dark red-flowered cultivar Roter Vogel some white-flowered plants appeared as the result of a mutation of the genetic factor Anl involved in anthocyanin synthesis. The white flowers of these plants had red spots owing to back-mutations in the dermal cells of the young corolla.Owing to a striking unstability of the new allele of Anl, a number of mutants originated which differ mutually in the frequency of reversion, which expressed itself in the very substantial differences in the spot density of the limb of the corolla. Between a mean number of less than one spot per cm² of the limb and a mean number of over 10.000 spots/cm², a series of transitions was found.The reversions did not remain restricted to the young epidermis but also occurred in sporogenous tissues. This resulted in the appearance of selfcoloured red descendants of plants with red-spotted white flowers. There is a positive correlation between the spot density of the parent plants and the percentage of plants with completely red corollas.The red spots on the corolla usually have the same colour as the wild type (Roter Vogel), but occasionally mutants occur with paler spots, the colour varying from a very pale pink to a red nearly as deep as in the wild type. The selfcoloured descendants of such mutants also show this colour variation from pale pink to red.On the grounds of these observations a theory was formulated which postulates that the Anl locus consists of a structural gene responsible for an enzyme active during anthocyanin synthesis and a regulatory element built up from intermediate repetitive DNA. This regulatory element in turn is built up of two components, one of which, the mutator, decides the activation of the structural gene while the other, the expressor, modifies the rate of activation. The mutations must be considered representative of larger or smaller deletions within one or both of these components. Reversions are the result of the restoration of the deletions by means of an amplification of the repetitive DNA in dividing cells of the developing flower buds.     

Locating the petunia Rf gene on a 650-kb DNA fragment

 A bulked segregant analysis was conducted in order to find RAPD and AFLP markers linked to the restorer of fertility (Rf ) gene in petunia. One RAPD marker, OP704, and one AFLP marker, ECCA/ MACT, were found to be closely linked to Rf (<1 cM) in our mapping population produced from an intraspecific Petunia hybrida cross. These two single-copy markers bracketing Rf were then mapped as RFLPs on the tomato map. Despite some rearrangement between the petunia and the tomato genomes, this synteny survey revealed two tomato markers, TG250 and CT24, closely linked to Rf. Physical mapping indicates that CT24, OP704 and ECCA/MACT lie on the same 650-kb MluI fragment. A physical to genetic distance ratio of 400 kb/cM around the Rf gene should make it feasible to identify markers physically very close to Rf. Received: 20 August 1997 / Accepted: 21 October 1997     

Conservation of alternative splicing and genomic organization of the myosin alkali light-chain (Mlc1) gene among Drosophila species

The Mlc1 gene of Drosophila melanogaster encodes two MLC1 isoforms via developmentally regulated alternative pre-mRNA splicing. In larval muscle and tubular and abdominal muscles of adults, all of the six exons are included in the spliced mRNA, whereas, in the fibrillar indirect flight muscle of adult, exon 5 is excluded from the mRNA. We show that this tissue-specific pattern of alternative splicing of the Mlc1 pre-mRNA is conserved in D. simulans, D. pseudoobscura, and D. virilis. Isolation and sequencing of the Mlc1 genes from these three other Drosophila species have revealed that the overall organization of the genes is identical and that the genes have maintained a very high level of sequence identity within the coding region. Pairwise amino acid identities are 94%-99%, and there are no charge changes among the proteins. Total nucleotide divergence within the coding region of the four genes supports the accepted genealogy of these species, but the data indicate a significantly higher rate of amino acid replacement in the branch leading to D. pseudoobscura. A comparison of nucleotide substitutions in the coding portions of exon 5 and exon 6, which encode the alternative carboxyl termini of the two MLC1 isoforms, suggests that exon 5 is subject to greater evolutionary constraints than is exon 6. In addition to the coding sequences, there is significant sequence conservation within the 5' and 3' noncoding DNA and two of the introns, including one that flanks exon 5. These regions are candidates for cis- regulatory elements. Our results suggest that evolutionary constraints are acting on both the coding and noncoding sequences of the Mlc1 gene to maintain proper expression and function of the two MLC1 polypeptides.      

Genetic control of dihydroflavonol 4-reductase gene expression in Petunia hybrida

The functions of four loci ( An1, An2, An4 , and An6 ) which control pigmentation in flowers of Petunia hybrida have been characterized. Linkage-analysis and molecular complementation experiments showed that the An6 locus contains the structural dfrA gene, encoding the enzyme dihydroflavonol 4-reductase (DFR). Analysis of gus gene expression driven by the dfrA promoter in transgenic plants showed that the dfrA promoter is highly active in the flower corolla, the anthers and seeds and, at a lower level, in ovules and the flower stem. These data are discussed in relation to the expression of other pigmentation genes and the accumulation pattern of anthocyanins. The expression of the dfrA-gus transgene was dependent on the genes an1 (in every tissue), an2 (in the flower limb only) and an4 (in anthers), demonstrating that these genes encode regulatory factors that control dfrA promoter activity.     

Role of interleukin-7 in degenerative and inflammatory joint diseases

IL-7 is known foremost for its immunostimulatory capacities, including potent T cell-dependent catabolic effects on bone. In joint diseases like rheumatoid arthritis and osteoarthritis, IL-7, via immune activation, can induce joint destruction. Now it has been demonstrated that increased IL-7 levels are produced by human articular chondrocytes of older individuals and osteoarthritis patients. IL-7 stimulates production of proteases by IL-7 receptor-expressing chondrocytes and enhances cartilage matrix degradation. This indicates that IL-7, indirectly via immune activation, but also by a direct action on cartilage, contributes to joint destruction in rheumatic diseases.