E. Veltkamp

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.

Regulation of flavonoid gene expression in Petunia hybrida: Description and partial characterization of a conditional mutant in chalcone synthase gene expression

SummaryWhite flowers of the Petunia hybrida line W43 accumulate glucosides of 4-coumaric acid and caffeic acid and are able to synthesize anthocyanins from exogeneously supplied naringenin, suggesting that W43 is blocked in a biosynthetic step preceding the formation of naringenin. The cultivar Red Star contains a similar mutation as W43; the genetic background of this cultivar, however, allows the production of considerable amounts of anthocyanins in certain areas of the flower. When grown at reduced light, flowers of Red Star are uniformly coloured, whereas under an enhanced light regime the flowers exhibit alternating white and coloured areas.In white sectors of flowers with a colour pattern virtually no chalcone synthase (CHS) enzyme activity could be demonstrated. The enzymes chalcone isomerase (CHI), UDPG: flavonoid-3-0-glucosyltransferase (3GT) and SAM: anthocyanin methyltransferase (OMT) are present, although at a more or less reduced level. From Western blotting and in vitro translation experiments we infer that the absence of CHS enzyme activity in the white sectors of the cultivar Red Star is due to the absence of translationally active CHS mRNA. The potential use of the mutants for genetic engineering in plants is emphasized.

Maintenance of multicopy plasmid Clo DF13 in E. coli cells: Evidence for site-specific recombination at parB

Certain derivatives of copy-control mutants of plasmid Clo DF13 are not stably inherited in E. coli. These plasmids, predominantly present as multimeric DNA molecules, lack a specific region, designated parB. Here we present the nucleotide sequence of this parB region spanning 328 bp between 46% and 49% on the plasmid genome. parB is a noncoding region with extensive internal symmetry. A recA-independent, site-specific resolution process occurs between two intramolecular parB sites present in direct orientation relative to each other. A gene located in the direct vicinity of parB, gene L, is not essential for parB functioning. However, our genetic data indicate that transcription from the gene L-containing operon into parB is required. We conclude that the efficient maintenance of Clo DF13 cop derivatives containing parB is provided by resolution of mutimeric molecules. Because Clo DF13 wt and cop derivatives have a different response to the deletion of parB we postulate that two different recombination systems, a parB-dependent and a parB-independent system, operate in the efficient maintenance of Clo DF13 plasmids.

Protein H encoded by plasmid Clo DF13 involved in lysis of the bacterial host

SummaryThe gene expression of the Clo DF13 “replication region”, located between 1.8% and 12% on the plasmid genome, was studied using newly constructed Clo DF13 insertion and deletion mutants. We were able to detect a Clo DF13 specified protein of 6 kilodaltons (kd) by electrophoretic analysis of plasmid proteins, synthesized in Escherichia coli minicells, on 14–25% gradient polyacrylamide gels. The gene encoding this protein was mapped between 1.8% and 12% on the Clo DF13 genome. The nucleotide sequence of this region, as determined by Stuitje et al. (1980), revealed three open reading frames each potentially coding for a protein of 6 kd. Since these three proteins differ in amino acid composition we could distinguish which of these proteins was actually synthesized, by labeling Clo DF13 proteins with specific 14C-labeled amino acids. We found that gene H, located between 9.3% (bp 744) and 11% (bp 893), encodes the observed protein of 6 kd (denominated protein H). With respect to the subcellular localization we observed that protein H, which contains a large hydrophobic region at its C-terminal part, is predominantly present in the bacterial membrane. Although gene H is located close to the region known to be involved in Clo DF13 replication, its gene product, protein H, is not essential for the plasmid DNA replication process. The possibility of the existence of a comparable protein encoded by the related plasmid Col E1 will be discussed.

Maintenance of the bacteriocinogenic plasmid Clo DF13 in Escherichia coli cells. II. Specific recombination functions involved in plasmid maintenance.

SummaryClo DF13 plasmids that are present at high copy-number in bacterial cells, such as Clo DF13 cop1 Ts, cop2 and cop3 are not stably inherited in the progeny, when certain plasmid DNA regions have been deleted. We have localized two Clo DF13 DNA regions involved in stable maintenance through accurate partitioning (par) namely parA, located between 71% and 72% and parB, located between 45% and 50% on the Clo DF13 genome. The instability of these cop plasmids which is accompanied by the formation of high amounts of multimeric DNA molecules, could be abolished by the insertion of transposon Tn901 into the plasmid genome. In particular that part of Tn901, that encodes for the site-specific recombination/ resolution system, appeared to be essential for stabilizing plasmid molecules. Wild-type parA- and/or parB- Clo DF13 plasmids, in contrast to cop mutants lacking these regions, are stably maintained during subsequent cell division, indicating that other (host specified) functions contribute to plasmid stability. Analysis of the role of host recombination systems in plasmid partitioning revealed that the recA function has no influence and recBC contributes only weakly to plasmid stability. With respect to the recE pathway, however, we found that in a recE proficient host all plasmids, even those lacking parA and/or parB, are stably maintained, indicating that the function of parA and parB can be replaced not only by the site-specific resolution functions of transposon Tn901, but also by the recE system. The possible role of plasmid specified and host specified functions in plasmid partitioning will be discussed.

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13

The complete nucleotide sequence of the bacteriocinogenic plasmid CloDF13 has been determined. The plasmid consists of 9957 base pairs (molecular weight 6.64 X 10(6] with a GC content of 54.4%. At this moment 16 identified biological functions can be assigned to the primary structure of the CloDF13 DNA. The functions include those of eight protein encoding genes, two untranslated RNA species, and six DNA sites. We discuss these functions in relation to the structure of CloDF13 DNA. For convenience we have divided the CloDF13 genome into five defined regions: region I (origin of vegetative replication, priming and control of replication, type I incompatibility), region II (cloacin DF13, cloacin immunity, cloacin release, cloacin operon control), region III (double-stranded DNA-phage interaction, type II incompatibility, multimer resolution), region IV (inhibition of male specific RNA phages and transfer of Flac), and region V (mobility proteins, basis of mobility).

Localization and nucleotide sequence of the bom region of Clo DF13

SummaryMobilization of the non-conjugative plasmid Clo DF13 requires both gene products of a conjugative plasmid and Clo DF13 encoded proteins as well as a cis-acting Clo DF13 DNA region, termed bom (basis of mobility). The bom region was located within a 264 bp fragment around the unique HpaI site. Comparison with the corresponding ColE1 and pBR322 sequences showed similarities with respect to the secondary structure. With respect to a possible relationship between the origin of vegetative replication (oriV) and the origin of transfer (oriT), we found that neither distance nor orientation of the Clo DF13 bom region with respect to oriV had any significant influence on the mobilization frequency. Surprisingly, after cloning of the 264 bp HpaII fragment in a bom- vector, restoration of the bom+ phenotype was only observed in one orientation. From these observations and from the sequence analysis of the bom region we suggest that transcription into this cloned bom fragment is essential for effective mobilization of the plasmid.

Origin and direction of replication of the bacteriocinogenic plasmid Clo DF13

Cairns type replicative intermediates of both the wildtype Clo DF13 plasmid and the copy mutant CLO DF13 cop3 were isolated by dye-buoyant density centrifugation. Replicative intermediates were linearized at the HpaI or Sa1I cleavage site, and examined with the electron-microscope. The data show that replication of both the Clo DF13 wild type plasmid and the Clo DF13 cop3 plasmid, initiates at about 2.8% on the physical map. Replication proceeds unindirectionally and counterclockwise on this map.

Maintenance of multicopy plasmid Clo DF13

SummaryTo elucidate the mechanisms that operate in plasmid maintenance, we analysed the stability of different combinations of Clo DF13 derivatives present in the same bacterial cell. From the data described in this paper we conclude: (i) each Clo DF13 plasmid molecule has an equal chance of colonizing daughter cells upon cell division, (ii) the Clo DF13 minimal replicon harbours functions involved in plasmid segregation and incompatibility, (iii) in the case of cells harbouring plasmid replicons which differ in size, the smaller plasmid is gradually lost from the cell population, (iv) in the case of cells habouring plasmid replicons which differ in copy number, the lower copy number plasmid is always lost from the cells population. The effect of plasmid copy number is dominant over the effect of plasmid size.

Stable Maintenance of Plasmid CLO DF13: Structural and Functional Relationships Between Replication Control, Partitioning, and Incompatibility

Nonconjugative plasmids are a very attractive model system for the study of the structural and functional aspects of stable maintenance of replicons in a growing bacterial population. Biochemical and molecular genetic studies have resulted in more insight into the genes and sites involved in the processes responsible for plasmid maintenance such as plasmid replication and its control, accurate partitioning of plasmids over daughter cells, and incompatibility between replicons.