The TACPyAT repeats in the chalcone synthase promoter of Petunia hybrida act as a dominant negative cis-acting module in the control of organ-specific expression.

Analysis of the expression of the GUS reporter gene driven by various regions of the Petunia hybrida chalcone synthase (chsA) promoter revealed that the developmental and organ-specific expression of the chsA gene is conferred by a TATA proximal module located between -67 and -53, previously designated as the TACPyAT repeats. Histochemical analysis of GUS reporter gene expression revealed that the organ-specific 67 bp promoter fragment directs the same cell-type specificity as a 530 bp promoter, whereas additional enhancer sequences are present within the more TATA distal region. Moreover, the region between -800 and -530 is also involved in extending the cell-type specificity to the trichomes of flower organs and of young seedlings. The mechanism by which the TACPyAT repeats modulate expression during plant development was studied by analysing the expression of the GUS gene driven by chimeric promoters consisting of the CaMV 35S enhancer (domain B, -750 to -90) fused to various chsA 5' upstream sequences. Detailed enzymatic and histochemical analysis revealed that in the presence of the TACPyAT module the CaMV 35S region only enhances GUS activity in those organs in which the chsA promoter is normally active. Furthermore, this analysis shows that enhancement in the presence of the CaMV 35S domain B is accomplished by increasing the number of cell types expressing the GUS gene within the organ, rather than enhancement of the chsA cell-type-specific expression within these organs. Deletion of the TACPyAT sequences in the chimeric promoter construct completely restores the well-documented CaMV 35S domain B cell-type specificity, showing that the TACPyAT module acts as a dominant negative cis-acting element which controls both organ and developmental regulation of the chsA promoter activity.

Promoter analysis of the chalcone synthase (chsA) gene of Petunia hybrida: a 67 bp promoter region directs flower-specific expression.

In order to scan the 5' flanking region of the chalcone synthase (chs A) gene for regulatory sequences involved in directing flower-specific and UV-inducible expression, a chimaeric gene was constructed containing the chs A promoter of Petunia hybrida (V30), the chloramphenicol acetyl transferase (cat) structural sequence as a reporter gene and the chs A terminator region of Petunia hybrida (V30). This chimaeric gene and 5' end deletions thereof were introduced into Petunia plants with the help of Ti plasmid-derived plant vectors and CAT activity was measured. A 220 bp chs A promoter fragment contains cis-acting elements conferring flower-specific and UV-inducible expression. A promoter fragment from -67 to +1, although at a low level, was still able to direct flower-specific expression but could not drive UV-inducible expression in transgenic Petunia seedlings. Molecular analysis of binding of flower nuclear proteins to chs A promoter fragments by gel retardation assays showed strong specific binding to the sequences from -142 to +81. Promoter sequence comparison of chs genes from other plant species, combined with the deletion analysis and gel retardation assays, strongly suggests the involvement of the TACPyAT repeats (-59 and -52) in the regulation of organ-specificity of the chs A gene in Petunia hybrida. We also describe an in vitro organ-specific transient expression system, in which flower or purple callus protoplasts are used, that enables us to pre-screen organ-specific expression of a chimaeric reporter gene.

Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida.

Chalcone synthase-encoding genes (chs) in Petunia hybrida comprise a multigene family. Some of the chs genes have been grouped into a subfamily, based upon their strong cross-hybridization and tight genomic linkage. From genomic libraries eight 'complete' chs genes, two chs gene 5'-fragments and two chs gene 3'-fragments have been isolated. The nucleotide sequence of six complete chs genes is presented and discussed in relation to their evolutionary origin and expression in different tissues. Each member of the family consists of two exons separated by an intron of variable size and sequence, which is located at a conserved position. The chs gene fragments represent single exons. Homology between non-linked chs genes is approx. 80% at the DNA level and restricted to protein-coding sequences. Homology between subfamily members (which are tightly linked) is higher (90-99%) and extends into untranslated regions of the gene, strengthening the view that they arose by recent gene duplications. The chsD gene contains a mutated translation stop codon, suggesting that this is an inactive (pseudo)gene. None of the other members of the gene family exhibits characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must characteristics of a pseudogene, indicating that if gene inactivation has occurred during their evolution, it must have been a recent event. Homology at the protein level between some (expressed) chs genes is surprisingly low. The possibility that these genes encode proteins with slightly different enzymatic activities is discussed.

The chalcone synthase multigene family of Petunia hybrida (V30): differential, light-regulated expression during flower development and UV light induction.

We have analysed the expression of the 8-10 members of the gene family encoding the flavonoid biosynthetic enzyme chalcone synthase (CHS) from Petunia hybrida. During normal plant development only two members of the gene family (CHS-A and CHS-J) are expressed. Their expression is restricted to floral tissues mainly. About 90% of the total CHS mRNA pool is transcribed from CHS-A, wheares CHS-J delivers about 10% in flower corolla, tube and anthers. Expression of CHS-A and CHS-J during flower development is coordinated and (red) light-dependent. In young seedlings and cell suspension cultures expression of CHS-A and CHS-J can be induced with UV light. In addition to CHS-A and CHS-J, expression of another two CHS genes (CHS-B and CHS-G) is induced in young seedlings by UV light, albeit at a low level. In contrast to CHS genes from Leguminoseae, Petunia CHS genes are not inducible by phytopathogen-derived elicitors. Expression of CHS-A and CHS-J is reduced to a similar extent in a regulatory CHS mutant, Petunia hybrida Red Star, suggesting that both genes are regulated by the same trans-acting factors. Comparison of the promoter sequences of CHS-A and CHS-J reveals some striking homologies, which might represent cis-acting regulatory sequences.

Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes.

In this paper we report the isolation of cDNA clones encoding the flavonoid-biosynthetic enzyme chalcone flavanone isomerase (CHI) from Petunia hybrida. A nearly full size cDNA clone, isolated from a corolla-specific expression library, was characterized by sequence analysis. Using this CHI cDNA and the previously cloned flavonoid-specific chalcone synthase (CHS) cDNA we show that CHI and CHS genes are coordinately and tissue-specifically expressed in a developmental and light-regulated manner. Furthermore, coordinate induction of both mRNAs is observed after continuous irradiation of Petunia plantlets with UV light, probably as part of the plants UV defence mechanism. The two CHI genes, denoted A and B, were isolated from a genomic library of the Petunia inbred line V30. Both genes are transcriptionally active: gene A is transcribed in corolla, tube and UV-irradiated plantlets (1.0 kb mRNA), whereas gene B is only transcribed in immature anthers (1.0 kb mRNA). In combination with Southern blot analysis these data implicate the presence of two distinct non-allelic CHI genes in the genome of the P. hybrida line V30. Unexpectedly, mature anthers accumulate a 0.3 kb larger CHI RNA. This RNA is transcribed from CHI gene A and has a 0.3 kb 5' extension relative to the gene A transcript found in corolla tissue. Furthermore it is neither coordinately expressed with CHS mRNA nor UV inducible. Its biological function is still obscure, since no active CHI enzyme could be demonstrated in the same tissue.

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology, chromosomal localization and evolutionary aspects.

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.

The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology, chromosomal localization and evolutionary aspects.

Chalcone synthase (CHS) genes in Petunia hybrida comprise a multigene family containing at least 7 complete members in the strain Violet 30 (V30). Based on a high sequence homology in both coding and non-coding sequence, a number of CHS genes can be placed into two subfamilies. By restriction fragment length polymorphism (RFLP) analysis it was shown that both chromosomes II and V carry one of these subfamilies, in addition to the other CHS genes identified so far. Members of a subfamily were found to be closely linked genetically. Analysis of the Petunia species that contributed to the hybrid nature of P. hybrida (P. axillaris, P. parodii, P. inflata and P. violacea) shows that none of the CHS gene clusters is specific for either one of the parents and therefore did not arise as a consequence of the hybridization. The number of CHS genes within a subfamily varies considerably among these Petunia species. From this we infer that the CHS subfamilies arose from very recent gene duplications.

Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family.

Twenty independent, petal-specific chalcone synthase (CHS) cDNA clones have been isolated from Petunia hybrida variety Violet 30 (V30). Sequence analysis shows that the largest of these clones contains the entire coding sequence. Using this clone in Southern blot analysis reveals the presence of multiple CHS gene copies in the genome of Petunia hybrida V30. Hybridization and sequence analysis of the CHS cDNA clones shows that they are all copied from a single mRNA species. This indicates the presence of only one transcriptionally active CHS gene in petals. Finally we report the identification, cloning and partial characterization of this gene.

Identification of mutations affecting replication control of plasmid Clo DF13.

The bacteriocinogenic plasmid Clo DF13, originally isolated from Escherichia cloacae, is stably maintained in Escherichia coli to the extent of about 10 copies per cell. Its replication resembles that of many other small, multicopy plasmids; plasmid-encoded protein is not required but plasmid-specific genetic information is involved in regulation of replication as both conditional and nonconditional copy-number mutants of Clo DF13, and transcomplementable copy-number mutants of plasmid Col E1 have been described. The sequences essential for replication of Col E1 (refs 16, 17) and Clo DF13 (refs 18, 19) have been identified within a region surrounding the replication origin. Initiation of Col E1 replication is preceded by transcription of the origin region, providing the RNA primer at the origin. However, transcription in the opposite direction results in a small transcript of approximately 100 nucleotides (RNA-100) for both Col E1 (refs 21, 22) and Clo DF13 (ref. 23). Data suggest that Col E1 RNA-100 acts as a negative control element for the initiation of replication. We show here that single base transitions in the RNA-100 cistron of Clo DF13 can result in a nonconditional increase in plasmid copy-number. Also, sequence analysis has revealed that a specific base transition in a DNA region, apparently involved in both termination and initiation of transcription towards the replication origin, results in a thermosensitive plasmid copy-number.

Molecular structure of the immunity gene and immunity protein of the bacteriocinogenic plasmid Clo DF13.

The nucleotide sequence of the Clo DF13 DNA region comprising the immunity gene has been determined. We also elucidated the aminoacid sequence of the 40 N-terminal and 7 C-terminal aminoacids of the purified immunity protein. From analysis of the data obtained we were able to locate the immunity gene between 11.7 and 14.5% on the Clo DF13 map, and to determine the complete aminoacid sequence of the immunity protein. It was observed that the Clo DF13 immunity gene encodes an 85 aminoacid protein and is transcribed in the same direction as the cloacin gene. These experimental data support our model, presented elsewhere, which implicates that the cloacin and immunity genes of Clo DF13 are coordinately transcribed from the cloacin promoter. We also present DNA sequence data indicating that an extra ribosome binding site precedes the immunity gene on the polycistronic mRNA. This ribosome binding site might explain the fact that in cloacinogenic cells more immunity protein than cloacin is synthesized. The comparison of the complete aminoacid sequence of the Clo DF13 immunity protein, with the aminoacid sequence data of the purified, comparable Col E3 immunity protein revealed that both proteins have extensive homologies in primary and secondary structure, although they are exchangeable only to a low extent in vivo and in vitro. It was also observed that a lysine residue was modified in immunity protein isolated from excreted bacteriocin complexes.

Differential excision from DNA of the C-8 deoxyguanosine reaction products of N-hydroxy-2-aminofluorene and N-acetoxy-N-acetyl-2-aminofluorene by endonuclease S1 from Aspergillus oryzae.

Calf thymus DNA was modified in vitro with [G-3H]N-hydroxy-2-aminofluorene and [G-3H]N-acetoxy-N-acetyl-2-aminofluorene and the nuclease S1 digestion was studied under identical conditions. The ratios of the maximum reaction rate (V) and the Michaelis constant (Km), V/Km, indicate that 2-aminofluorene(AF)-modified DNA is hydrolyzed 3 times more slowly than N-acetyl-2-aminofluorene(AAF)-modified DNA under similar reaction conditions. The AF-modified DNA was slightly more susceptible to partial digestion by nuclease S1 than unmodified control DNA. These results suggest that the local regions of denaturation induced by AF substitution are smaller than those associated with AAF modification.