Brassinosteroid-6-oxidases from Arabidopsis and tomato catalyze multiple C-6 oxidations in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via two parallel pathways, the early and late C-6 oxidation pathways according to the C-6 oxidation status. The tomato (Lycopersicon esculentum) Dwarf gene encodes a cytochrome P450 that has been shown to catalyze the C-6 oxidation of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis ortholog (AtBR6ox gene) of the tomato Dwarf gene. The encoded polypeptide has characteristics of P450s and is classified into the CYP85 family. The AtBR6ox and tomato Dwarf gene were expressed in yeast and the ability of the transformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both enzymes catalyze multiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro-6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol, and 6-deoxocastasterone to castasterone. Our results indicate that the AtBR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that these enzymes have a broad substrate specificity. This suggests that the BR biosynthetic pathway consists of a metabolic grid rather than two separate parallel pathways.

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis.

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.

Plants steroid hormones, brassinosteroids: current highlights of molecular aspects on their synthesis/metabolism, transport, perception and response.

Brassinosteroids (BRs) are plant steroids essential for normal growth and development and can be defined as steroids that carry an oxygen moiety at C-3 and additional ones at one or more of the C-2, C-6, C-22 and C-23 carbon atoms. BR biosynthesis and metabolism mutants have been obtained and the corresponding genes cloned. These include genes encoding 5alpha-reductase and cytochrome P450 enzymes, that are similar to enzymes associated with mammalian steroid synthesis. Perception and/or response mutants have also been identified via screening for altered sensitivity to BRs. Some of these mutants have been found to be defective in a leucine-rich repeat receptor kinase and in a component of a vacuolar ATPase. This review highlights the recent advances in unraveling BR synthesis/metabolism, transport, perception and response through the analysis of BR mutants.

The tomato DWARF enzyme catalyses C-6 oxidation in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are steroidal plant hormones essential for normal plant growth and development. Mutants in the biosynthesis or perception of BRs are usually dwarf. The tomato Dwarf gene (D), which was predicted to encode a cytochrome P450 enzyme (P450) with homology to other P450s involved in BR biosynthesis, was cloned previously. Here, we show that DWARF catalyses the C-6 oxidation of 6-deoxocastasterone (6-deoxoCS) to castasterone (CS), the immediate precursor of brassinolide. To do this, we first confirmed that the D cDNA complemented the mutant light- and dark-grown phenotypes of the extreme dwarf (dx) allele of tomato. To identify a substrate for the DWARF enzyme, exogenous application of BR intermediates to dx plants was carried out. C-6 oxoBR intermediates enhanced hypocotyl elongation whereas the C-6 deoxoBR, 6-deoxoCS, had little effect. Quantitative analysis of endogenous BR levels in tomato showed mainly the presence of 6-deoxoBRs. Furthermore, dx plants were found to lack CS and had a high level of 6-deoxoCS in comparison to D plants that had CS and a lower level of 6-deoxoCS. Confirmation that DWARF catalyzed the C-6 oxidation of 6-deoxoCS to CS was obtained by functional expression of DWARF in yeast. In these experiments, the intermediate 6alpha-hydroxycastasterone was identified, indicating that DWARF catalyzes two steps in BR biosynthesis. These data show that DWARF is involved in the C-6 oxidation in BR biosynthesis.

The tomato Dwarf gene isolated by heterologous transposon tagging encodes the first member of a new cytochrome P450 family.

To transposon tag the tomato Dwarf (D) gene, a tomato line that carries a T-DNA containing a maize Activator (Ac) transposable element closely linked to D was pollinated with a stock homozygous for the d mutation. Hybrid seedlings were screened for dwarf progeny, and three independent dwarf lines were obtained. Two of these lines showed inheritance of a recessive phenotype similar to that conferred by the extreme dwarf (dx) allele. Variegation for the dwarf phenotype in one of these lines suggested that D had been tagged by Ac. Genomic DNA adjacent to Ac in these two lines was isolated by use of the inverse polymerase chain reaction, and the two insertions mapped approximately 2 kb apart. Partial complementation of d was observed when the corresponding wild-type sequence was used in transformation experiments. A cDNA clone of D was sequenced, and the predicted amino acid sequence has homology to cytochrome P450 enzymes.

Germinal transpositions of the maize element Dissociation from T-DNA loci in tomato.

We have analyzed the pattern of germinal transpositions of artificial Dissociation (Ds) transposons in tomato. T-DNA constructs carrying Ds were transformed into tomato, and the elements were trans-activated by crossing to lines transformed with a stabilized Activator (sAc) that expressed the transposase gene. The sAc T-DNA carried a GUS gene to monitor its segregation. The Ds elements were inserted in a marker gene so that excision from the T-DNA could be monitored. The Ds elements also carried a genetic marker that was intended to be used for reinsertion selection of the elements after excision. Unfortunately, this gene was irreversibly inactivated on crossing to sAc. Germinal excision frequencies of Ds averaged 15-40%, but there was large variation between and within plants. Southern hybridization analysis of stable transposed Ds elements indicated that although unique transpositions predominate, early transposition events can lead to large clonal sectors in the germline of developing plants and to sibling offspring carrying the same transposition event. Multiple germinal transpositions from three different loci were examined for uniqueness, and 15 different transpositions were identified from each of three T-DNA loci that carried a single independent Ds. These were mapped relative to the donor T-DNA loci, and for each locus 70-80% of the transposed elements were closely linked to the donor site.

Variable expression of the herpes simplex virus thymidine kinase gene in Nicotiana tabacum affects negative selection.

The potentials and limitations of negative-selection systems based on the human herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which causes sensitivity to the nucleoside analog ganciclovir, were examined in tobacco as a model system. There were great differences between individual HSVtk(+) transgenic plants in ganciclovir sensitivity. Inhibition of growth while under selection correlated with HSVtk-tianscnpt levels. Negative selection against HSVtk(+) transformants at the level of Agrobacterium-mediated transformation using a ganciclo-vir/kanamycin double-selection medium (the positive selection marker neomycin phosphotransferase-II gene was in the transformation vector) resulted in a three- to six-fold reduction in the frequency of kanamycin-resistant shoots. The efficiency of negative selection in this case was limited due to the great variation in HSVtk expression, i.e., the frequently occurring transformants with low, or no, ganciclovir sensitivity escaping negative selection. Two independently constructed HSVtk genes showed the same variability of the phenotype in Nicotiana tabacum transformants. Distinct phenotypes, ranging from no regeneration through abnormal or delayed regeneration, were observed when leaf segments were placed on shoot-inducing medium supplemented with 10(-6)-10(-3) M ganciclovir. The highest HSVtk mRNA and ganciclovir sensitivity levels were observed in plants which were transformed with the pSLJ882 chimeric construct. The pSLJ882 plant expression vector carried the coding sequence of HSVtk, whereas plasmid pCX305.1 carried an HSVtk construct retaining the untranslated 5 leader and viral 3 regions. The pCX305.1 transformants showed, at most, a delayed formation of shoots with thin stems and very narrow leaves. Ganciclovir sensitivity showed typical Mendelian segregation. A gene-dosage effect was also seen at the seedling level in the progeny of two transgenic lines.

Analysis of the chromosomal distribution of transposon-carrying T-DNAs in tomato using the inverse polymerase chain reaction.

We are developing a system for isolating tomato genes by transposon mutagenesis. In maize and tobacco, the transposon Activator (Ac) transposes preferentially to genetically linked sites. To identify transposons linked to various target genes, we have determined the RFLP map locations of Ac- and Dissociation (Ds)-carrying T-DNAs in a number of transformants. T-DNA flanking sequences were isolated using the inverse polymerase chain reaction (IPCR) and located on the RFLP map of tomato. The authenticity of IPCR reaction products was tested by several criteria including nested primer amplification, DNA sequence analysis and PCR amplification of the corresponding insertion target sequences. We report the RFLP map locations of 37 transposon-carrying T-DNAs. We also report the map locations of nine transposed Ds elements. T-DNAs were identified on all chromosomes except chromosome 6. Our data revealed no apparent chromosomal preference for T-DNA integration events. Lines carrying transposons at known map locations have been established which should prove a useful resource for isolating tomato genes by transposon mutagenesis.

Interleukin 8 concentrations in amniotic fluid and peripheral venous plasma during human pregnancy and parturition.

To establish the gestational and labour-associated changes in interleukin 8 (IL-8) release, we have determined the concentration of this cytokine in maternal peripheral plasma and amniotic fluid from 15 weeks of gestation to term and in association with spontaneous-onset labour at term and preterm. No statistically significant changes in peripheral plasma IL-8 concentration were observed during pregnancy or in association with labour onset (mean concentration 56.5 +/- 14.5 ng/l, N = 64). The IL-8 concentrations in amniotic fluid were up to 50-fold greater than those observed in peripheral plasma (p < 0.05) and increased significantly (p < 0.05) during pregnancy. At term, but before the onset of labour, amniotic fluid concentrations of IL-8 averaged 969.2 +/- 553.5 ng/l (N = 12). In association with labour at term, IL-8 concentrations increased to 3895.8 +/- 1414.4 ng/l (N = 6, p < 0.03). The concentration of IL-8 in amniotic fluid obtained from women in preterm labour averaged 1854.7 +/- 1352.6 ng/l (N = 6) but was not statistically different from the concentration of IL-8 in amniotic fluid obtained from gestational aged-matched non-labouring controls. Although the precise role of intrauterine IL-8 at the time of parturition awaits elucidation, these data support the concept that this cytokine may be involved in the biochemical events associated with the onset and/or propagation of normal labour in the human.

Elevated plasma interleukin 6: a biochemical marker of human preterm labour.

Maternal peripheral venous plasma interleukin 6 (IL-6) concentrations were determined during human pregnancy and labour at term and preterm. During preterm labour, IL-6 concentrations were significantly elevated (p < 0.02) compared to gestationally matched, non-labouring controls (53.7 +/- 15.7 pg/ml, n = 17, and 15.4 +/- 6.4 pg/ml, n = 23, respectively). IL-6 concentrations did not vary significantly during normal pregnancy and labour at term. These data support a role for IL-6 in the pathogenesis of human preterm labour, are evidence for the contention that preterm labour is mechanistically distinct at a biochemical level from normal labour at term and identify maternal peripheral venous plasma IL-6 as a biochemical marker of this condition.

Effective vectors for transformation, expression of heterologous genes, and assaying transposon excision in transgenic plants.

Progress in plant molecular biology has depended heavily on the availability of effective vectors for plant cell transformation and heterologous expression. In this paper we describe the structures of a wide array of plasmids which have proved extremely effective in (a) plant transformation, (b) expression of heterologous genes and (c) assaying the activity of transposons in transgenic plants. Constructs that confer resistance to kanamycin, hygromycin, streptomycin, spectinomycin and phosphinotricin, or that confer beta-glucuronidase (GUS) gene expression are presented. Binary vector constructs that carry polylinkers of the pUC and Bluescript types are also described. Plasmids that permit the expression of any heterologous reading frame from either nopaline synthase (nos) or octopine synthase (ocs) promoters, as well as the cauliflower mosaic virus 35S promoter, using either the nopaline synthase or octopine synthase 3' polyadenylation sequences, are presented. These constructs permit a choice of orientation of the resulting transgene of interest, relative to the orientation of the selection marker gene. Most of the plasmids described here are publicly available.

Elevated maternal plasma immunoreactive phospholipase A2 in human preterm and term labour.

Peripheral plasma concentrations of immunoreactive phospholipase A2 (irPLA2) (Type II, non-pancreatic) were determined in 110 women during pregnancy. The concentration of irPLA2 did not significantly change during pregnancy (5.7 +/- 0.6 ng/ml, n = 72) until the onset of labour. When compared with non-labouring women, irPLA2 concentrations were significantly elevated in association with both preterm labour (13.3 +/- 2.4 ng/ml, n = 15, p less than 0.02) and labour at term (10.4 +/- 1.7, n = 23, p less than 0.02). These data suggest that maternal plasma irPLA2 may be reflective of the mechanism(s) underlying the labour-associated increase in human gestational tissue eicosanoid formation.

Laparoscopic follow-up of patients with ovarian carcinoma.

Laparoscopy with cytology of ascitic fluid or peritoneal washings was performed on 110 occasions in 62 patients with ovarian cancer to assess response to chemotherapy. Damage to bowel occurred on three occasions and complete visualisation of the peritoneal cavity was not possible in 14 patients. When tumour was seen and/or cytology was positive, the prognosis was poor; absence of macroscopic tumour with negative cytology did not preclude continuing disease. A group of patients was identified in whom a change of therapy based on laparoscopic findings after six months of treatment, might have proven beneficial. Laparoscopy has a limited place as a second-look procedure in patients undergoing treatment for carcinoma of the ovary.

Diagnostic vacuum curettage of the uterus.

A review of the literature on vacuum curettage is presented. The experience of vacuum curettage with the Vabra apparatus is illustrated by four cases which demonstrate the usefulness of this procedure. It is concluded that this procedure is a useful adjunct to gynaecological diagnosis.