Hirsutinolides,glaucolides and sesquiterpene lactone from Vernonia species

The investigation of nine Vernonia species afforded in addition to known sesquiterpenes 28 new ones. The structures were elucidated by high field ¹H NMR spectroscopy and the configurations were determined by NOE difference spectroscopy and, in one case, by X-ray analysis. The results indicated that configurations of several previously reported sesquiterpene lactones have had to be revised. In addition to known types two new ones, the jalcaguaianolides and the vernojalcanolides, are described. Furthermore some unusual reaction products are presented which, in part, led to some natural occurring lactones.     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and -glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.     

Agrobacterium mediated transformation of chickpea (Cicer arietinum L.) embryo axes

 Embryo axes of four accessions of chickpea (Cicer arietinum L.) were treated with Agrobacterium tumefaciens strains C58C1/GV2260 carrying the plasmid p35SGUSINT and EHA101 harbouring the plasmid pIBGUS. In both vectors the GUS gene is interrupted by an intron. After inoculation shoot formation was promoted on MS medium containing 0.5 mg/l BAP under a selection pressure of 100 mg/l kanamycin or 10 mg/l phosphinothricin, depending on the construct used for transformation. Expression of the chimeric GUS gene was confirmed by histochemical localization of GUS activity in regenerated shoots. Resistant shoots were grafted onto 5-day-old dark-grown seedlings, and mature plants could be recovered. T-DNA integration was confirmed by Southern analysis by random selection of putative transformants. The analysis of 4 plantlets of the T1 progeny revealed that none of them was GUS-positive, whereas the presence of the nptII gene could be detected by polymerase chain reaction. Received: 30 May 1997 / Revision received: 18 September 1997 / Accepted: 22 March 1999     

Ebstein’s anomaly may be caused by mutations in the sarcomere protein gene MYH7

Ebstein’s anomaly is a rare congenital heart malformation characterised by adherence of the septal and posterior leaflets of the tricuspid valve to the underlying myocardium. Associated abnormalities of left ventricular morphology and function including left ventricular noncompaction (LVNC) have been observed. An association between Ebstein’s anomaly with LVNC and mutations in the sarcomeric protein gene MYH7, encoding β-myosin heavy chain, has been shown by recent studies. This might represent a specific subtype of Ebstein’s anomaly with a Mendelian inheritance pattern. In this review we discuss the association of MYH7 mutations with Ebstein’s anomaly and LVNC and its implications for the clinical care for patients and their family members.     

Thidiazuron-induced plant regeneration from protoplasts of Vicia faba cv. Mythos

Summary Protoplasts of 10 cultivars of V. faba were isolated from etiolated shoot-tips and tested for their regeneration capacity. After purification, protoplasts were embedded in sodium alginate and cultivated in the medium of Kao and Michayluk (1975) containing 0.5 mg·1^–1 of each 2,4-dichlorophenoxyacetic acid, naphthylacetic acid and 6-benzylaminopurine. Depending on cultivar, division frequencies of up to 40% were obtained. Six weeks after embedding, protoplast-derived calluses were transferred to Gelrite-solidified media with different combinations of growth regulators. A two step protocol (auxin high/low) was tested for its ability to induce somatic embryogenesis. The formation of globular structures was observed, but no embryo formation could be achieved. In contrast, cultivation of protocalluses on medium supplemented with thidiazuron resulted in shoot development in cultivar Mythos. To generate mature plants, the shoots were grafted onto young seedlings. In order to optimize the in vitro-conditions, different concentrations of thidiazuron alone or in combination with naphthylacetic acid were tested, showing that an increase of thidiazuron and the addition of naphthylacetic acid positively affects both the viability of protocalluses and the regeneration frequency.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BA 5-benzylaminopurine - GA₃ gibberellic acid - IBA indole-3-butyric acid - Kin kinetin - KM Kao and Michayluk - MS Murashige and Skoog - NAA naphthylacetic acid - TDZ thidiazuron - Zea zeatin     

Additive effects of the feed-back insensitive bacterial aspartate kinase and the Brazil nut 2S albumin on the methionine content of transgenic narbon bean (Vicia narbonensis L.)

So far two different strategies for engineering high methionine (Met) grain legumes were followed separately in several laboratories: a) The transfer of foreign genes encoding Met-rich proteins, and b) the engineering of Met biosynthesis pathways. In some cases a down regulation of the formation of endogenous sulfur-containing compounds was observed due to the expression of Met-rich foreign proteins. Since this might result from competition of the foreign protein with endogenous compounds for limited Met supply both strategies were combined in the present work. Double transformants of narbon bean (Vicia narbonensis L.) were generated which express seed-specifically the Met-rich Brazil nut 2S albumin (BNA) as well as a feed-back insensitive bacterial aspartate kinase (AK) known to stimulate Met biosynthesis in transgenic tobacco seeds. In order to produce double transformants a homozygous transgenic BNA line of narbon bean was either retransformed with the AK gene or crossed with an AK line. For the first time the influence of a deregulated AK on amino acids of the aspartate pathway was studied in seeds of a transgenic legume. Effects of expressing the foreign genes on inorganic sulphate, free and protein-bound Met and other amino acids of the aspartate pathway as well as on free sulphhydryl compounds of mature seeds were analysed. AK lines had 10 to 12 percent and the BNA line 80 percent increased Met in mature seeds. Double transformants showed additive but not synergistic effects of the expression of AK and BNA gene on seed Met. In their mature seeds protein-bound Met reached levels 2.0 to 2.4 times higher than in the wildtype. The Met level of best line corresponds approximately to the FAO standard for Met in a nutritionally balanced protein for human food or for feeding monogastric animals.     

Agrobacterium-mediated transformation of Vicia faba

Among the major grain legume crops, Vicia faba belongs to those where the production of transgenic plants has not yet convincingly been reported. We have produced stably transformed lines of faba bean with an Agrobacterium tumefaciens-mediated gene transfer system. Stem segments from aseptically germinated seedlings were inoculated with A. tumefaciens strains EHA101 or EHA105, carrying binary vectors conferring (1) uidA, (2) a mutant lysC gene, coding for a bacterial aspartate kinase insensitive to feedback control by threonine, and (3) the coding sequence for a methionine-rich sunflower 2S-albumin, each in combination with nptII as selectable marker. Kanamycin-resistant calluses were obtained on callus initiation medium at a frequency of 10–30%. Shoot regeneration was achieved on thidiazuron containing medium in a second culture step. A subsequent transfer of shoots to BA-containing medium was necessary for stem elongation and leaf development. Shoots were rooted or grafted onto young seedlings in vitro and mature plants were recovered. Molecular analysis confirmed the integration of the transgenes into the plant genome. Inheritance and expression of the foreign genes was demonstrated by Southern blot, PCR, western analysis and enzyme activity assays. Although at present the system is time-consuming and of relatively low efficiency, it represents a feasible approach for the production of genetically engineered faba beans.     

A chimeric gene encoding the methionine-rich 2S albumin of the Brazil nut (Bertlrolletia excelsa H.B.K.) is stably expressed and inherited in transgenic grain legumes

The coding region of the 2S albumin gene of Brazil nut (Bertholletia excelsa H.B.K.) was completely synthesized, placed under control of the cauliflower mosaic virus (CaMV) 35S promoter and inserted into the binary vector plasmid pGSGLUC1, thus giving rise to pGSGLUC1-2S. This was used for transformation of tobacco (Nicotiana tabacum L. cv. Petit Havanna) and of the grain legume Vicia narbonensis L., mediated by the supervirulent Agrobacterium tumefaciens strain EHA 101. Putative transformants were selected by screening for neomycin phosphotransferase (NPT II) and β-glucuronidase (GUS) activities. Transgenic plants were grown until flowering and fruiting occurred. The presence of the foreign gene was confirmed by Southern analysis. GUS activity was found in all organs of the regenerated transgenic tobacco and legume plants, including the seeds. In the legume, the highest expression levels of the CaMV 35S promoter-controlled 2S albumin gene were observed in leaves and roots. 2S albumin was localized in the vacuoles of leaf mesophyll cells of transgenic tobacco. The Brazil nut protein was present in the 2S fraction after gel filtration chromatography of the legume seed proteins and could be clearly identified by immunoblotting. Analysis of seeds from the R₂ progenies of the legume and of transgenic tobacco plants revealed Mendelian inheritance of the foreign gene. Agrobacterium rhizogenes strain RifR 15834 harbouring the binary vector pGSGLUCl2S was also used to transform Pisum sativum L. and Vicia faba L. Hairy roots expressed the 2S albumin-specific gene. Several shoots were raised but they never completely rooted and no fertile plants were obtained from these transformants.