Distinction between cultivated and wild chicory gene pools using AFLP markers.

The cultivation area of industrial chicory, Cichorium intybus L. cv Sativum, coincides with the natural distribution area of its wild relative, C. intybus L., which could lead to gene flow between wild and cultivated types. The genetic diversity within and between the two types has therefore been studied using AFLP genotyping of samples from 12 wild populations collected in Belgium and ten commercial varieties. The genotyping of 233 individuals allowed the identification of 254 AFLP markers. Similar levels of genetic diversity were observed within wild populations and cultivated varieties, suggesting the absence of any strong bottleneck in the history of the cultivated types. The phylogenetic analysis pointed to a monophyletic origin of cultivated varieties as compared to the local wild populations studied, hence the two types of chicory form two separate gene pools. The genotyping of some individuals sampled in ruderal sites clearly showed that they belong to the cultivated gene pool, which suggests the existence of feral or weedy types. The low differentiation observed among wild populations indicates that gene flow might be important in this species.

Potato tubers exhibit both homolytic and heterolytic hydroperoxide fatty acid-cleaving activities.

The action of a crude potato-tuber extract on 9- and 13-hydroperoxides of linoleic and linolenic acids was investigated. HPLC analysis revealed that 50% of the 9-hydroperoxide isomers and almost all the 13-hydroperoxide isomers were rapidly enzymically metabolized. No degradation of fatty acid hydroperoxides was observed with a thermally denatured enzymic extract. GC-MS identification of the volatiles formed by the reaction revealed that no volatiles were detected from the 9-hydroperoxide isomers, whereas 13-hydroperoxide of linolenic acid was cleaved into (Z)-3-hexenal, pentenols or dimers of pentene.

Molecular cloning and characterization of the enzyme UDP-glucose: protein transglucosylase from potatodaggerdaggerThis paper is specially dedicated to the memory of Dr Juana S. Tandecarz, deceased on December 10, 1996.

UDP-Glc:protein transglucosylase (UPTG) (EC 2.4.1.112) is an autocatalytic glycosyl-transferase previously postulated as a protein that primes starch biosynthesis. Polyclonal antibodies raised against UPTG purified from potato (Solanum tuberosum L.) tubers were used to screen a potato swelling stolon tip cDNA expression library. The isolation, cloning and sequencing of two cDNAs corresponding to UPTG are described. Recombinant UPTG was labelled after incubation with UDP-[(14)C]-Glc and Mn(2+), indicating that it was enzymatically active. It was determined that purified as well as recombinant UPTG can be reversibly glycosylated by UDP-Glc, UDP-Xyl or UDP-Gal. RNA hybridization studies and western blot analysis indicate that UPTG mRNA and protein are expressed in all potato tissues. Databank searches revealed a high degree of identity between UPTG and several plant sequences that encode for proteins with apparent localization at the cytoplasmic face of the Golgi apparatus and at plasmodesmata. The biochemical properties of UPTG and the apparent lack of a signal peptide that could allow its entrance into plastids argue against the postulated role of UPTG in starch synthesis and point towards a possible role of the protein in the synthesis of cell wall polysaccharides.

Identification of cytosolic Mg2+-dependent soluble inorganic pyrophosphatases in potato and phylogenetic analysis.

Using polyclonal antibodies raised against a previously cloned potato Mg2+-dependent soluble inorganic pyrophosphatase (ppa1 gene) [8], a second gene, called ppa2, could be isolated. A single locus homologous to ppa2 was mapped on potato chromosomes, unlinked to the two loci identified for ppa1. From a phylogenetic and structural point of view, the PPA1 and PPA2 polypeptides are more closely related to prokaryotic than to eukaryotic Mg2+-dependent soluble inorganic pyrophosphatases (soluble PPases). Subcellular localization by immunogold electron microscopy, using sections from leaf parenchyma cells, showed that PPA and PPA2 are localized to the cytosol. Based on these observations, the likely phylogenetic origin and the physiological significance of the cytosolic soluble pyrophosphatases are discussed.

Comparison between plain and gentamicin containing collagen sponges in infected peritoneal cavity in rats.

OBJECTIVE: To study the usefulness of gentamicin-containing sponges in the infected peritoneal cavity in rats. DESIGN: Controlled study. MATERIAL: 83 Male Wistar rats, 36 of which were treated by plain sponge, 36 by gentamicin-impregnated sponge, and 11 acted as controls. INTERVENTIONS: A standard model of intraperitoneal infection was developed by making a 1 cm long incision in the caecum. MAIN OUTCOME MEASURES: Postoperative mortality, macroscopic and microscopic features of infection, and bacterial concentrations in the abdomen on days 3, 6, and 9. RESULTS: In the respective groups 2, 3 and 2 animals died during operation and 4/34 (11%), 3/33 (9%); and 0/9 died before day 3. There were appreciable reductions in the number of animals with features of infection after 3 days but these were not significant at 6 or 9 days. CONCLUSION: Gentamicin-containing collagen sponges placed on a septic focus in the abdomen reduce local infection for at least 3 days.

Molecular cloning and characterization of a soluble inorganic pyrophosphatase in potato.

A cDNA clone encoding a soluble inorganic pyrophosphatase (EC 3.6.1.1) of potato (Solanum tuberosum L.) was isolated by screening a developing tuber library with a heterologous probe. The central domain of the encoded polypeptide is nearly identical at the sequence level with its Arabidopsis homolog (J.J. Kieber and E.R. Signer [1991] Plant Mol Biol 16: 345-348). Computer-assisted analysis of the potato, Arabidopsis, and Escherichia coli soluble pyrophosphatases indicated a remarkably conserved organization of the hydrophobic protein domains. The enzymatic function of the potato protein could be deduced from the presence of amino acid residues highly conserved in soluble pyrophosphatases and was confirmed by its capacity to complement a thermosensitive pyrophosphatase mutation in E. coli. The potato polypeptide was purified from complemented bacterial cells and its pyrophosphatase activity was shown to be strictly dependent on Mg2+ and strongly inhibited by Ca2+. The subcellular location of the potato pyrophosphatase is unknown. Structure analysis of the N-terminal protein domain failed to recognize typical transit peptides and the calculated molecular mass of the polypeptide (24 kD) is significantly inferior to the values reported for the plastidic (alkaline) or mitochondrial pyrophosphatases in plants (28-42 kD). Two unlinked loci could be mapped by restriction fragment length polymorphism analysis in the potato genome using the full-length cDNA as probe.

Expression of intron-encoded maturase-like polypeptides in potato chloroplasts.

The trnK gene has been identified on a cloned plastid DNA fragment of potato (Solanum tuberosum cv Désirée). This gene codes for a tRNA-Lys and is interrupted by a 2.5-kb intron belonging to the group II organellar introns. In addition, this intervening sequence contains a long open reading frame potentially coding for a 509 amino-acid polypeptide (ORF509) related to mitochondrial intron-encoded maturases from fungi. The translational capacity of the trnK intron was first demonstrated in vitro in a prokaryotic DNA-directed expression system. In order to examine the expression of the intron in the potato plant, a synthetic peptide corresponding to the last nine amino acids of the predicted ORF509 product was used to raise antibodies. Western-blot experiments on chloroplast protein extracts, using a sensitive chemiluminescent detection system, identified polypeptides similar to in-vitro products. These results suggest that the trnK intron is expressed at the protein level in the plant. This is the first report of the in-vivo expression of an intron-encoded polypeptide in higher plant plastids.

Use of chloroplast DNA polymorphisms for the phylogenetic study of seven Phaseolus taxa including P. vulgaris and P. coccineus.

The genetic variability of seven Phaseolus taxa has been evaluated on the basis of molecular data and the results have used to clarify the phyletic relationships between several taxa of the P. coccineus L. complex. Chloroplast DNA (cpDNA) from 33 populations was digested with six restriction endonucleases, revealing some polymorphisms that made it possible to divide most of the taxa into two main groups: the subspecies of P. coccineus on the one hand, and P. vulgaris L., P. polyanthus Greenman and P. costaricensis (Freytag and Debouck) on the other hand. P. polyanthus is closer to P. vulgaris than the other taxa of the second group and should be considered as a separate species. The position of the wild species P. costaricensis is intermediate between P. coccineus and P. polyanthus. P. glabellus shows sufficient polymorphisms at the cpDNA level to be recognized as a separate species, as previously suggested from total seed-protein electrophoretic studies. These results favour the hypothesis of a common phylogeny for P. vulgaris, P. polyanthus, P. costaricensis and P. coccineus from a single wild ancestor. Although cpDNA is generally known to be uniform at the intraspecific level, some additional polymorphisms were also detected within P. vulgaris, P. polyanthus and P. coccineus. Further studies are required to understand the significance of the latter.

Homologies to plastid DNA in the nuclear and mitochondrial genomes of potato.

Potato plastid DNA clones, representing onefourth of the potato plastome complexity and containing sequences of the 16SrRNA, rps16, atpA, atpE, psaA, psaB, trnK, trnV, and trnG genes, were used as hybridization probes on nuclear- and mitochondrial-enriched DNAs. Each probe hybridized to multiple nuclear restriction fragments distinct from the plastid cleavage products generated by the same endonucleases. The nuclear hybridizable fragments are highly methylated at their Hpall target sequences (C/CGG). In some instances, the transfer seemed to involve plastid regions of several kilobase pairs, as reflected by the co-integration in the nucleus of restriction sites that are distant in the plastome. Three clones hybridized additionally to distinct mitochondrial fragments. These results indicate that extensive DNA transfers did occur between plastids and other organelles in potato.