Cell type matters: competence for alkaloid metabolism differs in two seed-derived cell strains of Catharanthus roseus

Protoplasma - Since the discovery of the anticancer drugs vinblastine and vincristine, Catharanthus roseus has been intensively studied for biosynthesis of several terpene indole alkaloids (TIAs)....     

Bioassembly of acyl lipids in microspore-derived embryos of Brassica campestris L.

The native lipid composition and the capacity of cell-free extracts to biosynthesize acyl lipids in vitro were determined for the first time using the recently reported microspore-derived (MD) embryo system from the Brassica campestris low erucic acid line BC-2 (Baillie et al. 1992). The total lipid fraction isolated from midcotyledonary stage MD embryos (21 days in culture) was composed primarily of triacylglycerol (76%) with an acyl composition quite similar to that of mature BC-2 seed. When incubated in the presence of glycerol-3-phosphate, ¹⁴C 181-CoA, and reducing equivalents, homogenates prepared from 21-day cultured MD embryos were able to biosynthesize glycerolipids via the Kennedy pathway. The maximum in vitro rate of triacylglycerol biosynthesis could more than account for the known rate of lipid accumulation in vivo. The homogenate catalyzed the desaturation of 181 to 182 and to a lesser extent, 183. The newly-synthesized polyunsaturated fatty acids initially accumulated in the polar lipid fraction (primarily phosphatidic acid and phosphatidylcholine) but began to appear in the triacylglycerol fraction after longer incubation periods. As expected for a low erucic acid cultivar, homogenates of MD embryos from the BC-2 line were incapable of biosynthesizing very long chain monounsaturated fatty acyl moieties (201 and 221) from 181-CoA in vitro. Nonetheless, embryo extracts were still capable of incorporating these fatty acyl moieties into triacylglycerols when supplied with ¹⁴C 201-CoA or ¹⁴C 221-CoA. Collectively, the data suggest that developing BC-2 MD embryos constitute an excellent experimental system for studying pathways for glycerolipid bioassembly and the manipulation of this process in B. campestris.Abbreviations CPT sn-1,2-diacylglycerol cholinephosphotransferase - DAG diacylglycerol - DGAT diacylglycerol acyltransferase - DGDG digalactosyldiacylglycerol - G-3-P glycerol-3-phosphate - G-3-PAT glycerol-3-phosphate acyltransferase - LPA lyso-phosphatidic acid - LPAT lyso-phosphatidic acid acyltransferase - LPC lyso-phosphatidylcholine - LPCAT acyl-CoA: lyso-phosphatidylcholine acyltransferase - LPE lyso-phosphatidylethanolamine - MGDG monogalactosyldiacylglycerol - PA phosphatidic acid - PA Phosphatase, phosphatidic acid phosphatase - PC phosphatidylcholine - PE phosphatidylethanolamine - PG phosphatidylglycerol - TAG triacylglycerol - 181-CoA oleoyl-Coenzyme A - 181 oleic acid, cis-9-octadecenoic acid - 182 linoleic acid, cis-9,12-octadecadienoic acid - 183 -linolenic acid, cis-9,12,15-octadecatrienoic acid - 201 cis-11-eicosenoic acid - 221 erucic acid, cis-13-docosenoic acid; all other fatty acids are designated by number of carbon atoms: number of double bonds National Research Council of Canada Publication No. 35896     

Production and characterization of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus

Summary Cell suspension-derived protoplasts of a chlorsulfuron-resistant (GH50) strain of Arabidopsis thaliana cv Columbia were X-irradiated at 60 or 90 krad, to facilitate the elimination of GH50 donor chromosomes in fusion products. Irradiated GH50 protoplasts were fused, with polyethylene glycol, to protoplasts derived from stem epidermal strips of Brassica napus cv Westar. Chlorsulfuron-resistant colonies were selected in vitro and then transferred to shoot and root regeneration medium. Seventeen hybrid lines were regenerated in vitro, and eight were successfully established in the greenhouse, where they flowered. These eight asymmetric hybrids were intermediate in vegetative morphology between Arabidopsis and Brassica. The flowers from these hybrids were male-sterile with abnormal petal and pistil structures. Zymograms for phosphoglucomutase, esterase, and peroxidase showed the presence of all parental isozymes in each of the hybrids tested. Nuclear hybridity was also confirmed for the ribosomal RNA genes using a wheat rDNA probe; however, the chloroplast genome in each of the hybrids was derived solely from the Brassica parent. All selected somatic hybrids were capable of rooting at levels of chlorsulfuron which were inhibitory to unfused Brassica plantlets. The degree of herbicide resistance in the hybrid shoots is presently being evaluated.Contribution No. 1428, Plant Research Centre, Agriculture Canada     

Non-random chloroplast segregation inNicotiana tabacum (+)N. rustica somatic hybrids selected by dual nuclear-encoded resistance

Nicotiana tabacum (+)N. rustica interspecific somatic hybrids were produced by fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistantNicotiana tabacum L. with leaf mesophyll protoplasts of transgenic kanamycin-resistantN. rustica L. Somatic hybrids were selected on the basis of resistance to both methotrexate and kanamycin. Evidence for nuclear hybridization was obtained for 21 hybrids by restriction-fragment-length-polymorphism (RFLP) analysis using a heterologous wheat nuclear ribosomal-DNA (rDNA) probe and by analysis of glutamate-oxaloacetate transaminase (GOT) isoenzymes. Chloroplasts segregated non-randomly as 20 of the somatic hybrids possessedN. rustica chloroplasts and only one hadN. tabacum chloroplasts. Patterns of mitochondrial inheritance were examined by hybridization of a heterologous wheat cytochrome oxidase subunit II (coxII) gene with genomic DNA of the somatic hybrids. Four somatic hybrids with hybridization patterns similar toN. rustica and 17 with hybridization patterns consistent with mitochondrial DNA (mtDNA) rearrangement or recombination were obtained. None of the somatic hybrids had patterns ofcoxll hybridization identical withN. tabacum. Male-fertility levels in the hybrids ranged from undetectable to 87% and only nine hybrids produced a limited amount of viable seed. There was no apparent correlation between the patterns of organelle inheritance in the somatic hybrids and the relative degree of fertility.Contribution No. 1439 Plant Research CentreCurrent address: Plant Biotechnology Institute, National Research Council, 110 Gymmasium Road, Saskatoon, Saskatchewan S7N OW9, Canada     

Effects of gene dosage and sequence modification on the frequency and timing of transposition of the maize element Activator (Ac) in tobacco

The effect of Ac copy number on the frequency and timing of germinal transposition in tobacco was investigated using the streptomycin phosphotransferase gene (SPT) as an excision marker. The activity of one and two copies of the element was compared by selecting heterozygous and homozygous progeny of transformants carrying single SPT::Ac inserts. It was observed that increasing gene copy not only increases the transposition frequency, but also occasionally alters the timing of transposition such that earlier events are obtained. The result is that some homozygous plants generate multiple streptomycin resistant progeny carrying the same transposed Ac (trAc) element. We have also investigated the effect of modification of the sequence in the region around 82 bp downstream of the polyadenylation site and 177 bp from the 3 end of the element on germinal excision frequencies. Alteration of three bases to create a BglII site at this location caused a minor decrease in germinal excision events, but insertion of four bases to create a Cla I site caused a 10-fold decrease in the transposition activity of the Ac element.     

Amino acid changes in the fourth conserved region of human immunodeficiency virus type 2 strain HIV-2ROD envelope glycoprotein modulate fusion.

The fourth conserved region (C4) of human immunodeficiency virus type 1 (HIV-1) surface glycoprotein has been shown to participate in CD4 binding and to influence viral tropism (A. Cordonnier, L. Montagnier, and M. Emerman, Nature [London] 340:571-574, 1989). To define the role of the corresponding region of HIV-2, we introduce single amino acid changes into the C4 sequence of HIV-2ROD. The effects of these mutations on glycoprotein function and on virus infectivity have been examined. We have shown that the tryptophan residue at position 428 is necessary primarily for CD4 binding. The isoleucine residue at position 421 is necessary for the establishment of productive infection in the promonocytic cell line U937, while it is dispensable to some extent for infection of primary T lymphocytes or the lymphocytic cell line SUP-T1. This replication defect correlated with the failure of the Ile-421-to-Thr (Ile-421-->Thr) mutant glycoprotein to form syncytia in U937 cells. DNA analysis of revertant viruses revealed that a strong selective pressure was exerted on residue 421 of the surface glycoprotein to allow HIV-2 infection of U937 cells. These results demonstrate that this region of HIV-2 plays an important role in determining fusion efficiency in a cell-dependent manner and consequently can influence viral tropism.     

Vascular expression of the grp1.8 promoter is controlled by three specific regulatory elements and one unspecific activating sequence

The bean grp1.8 full-length promoter is specifically active in vascular tissue during normal development of tobacco. Deletion of a negative regulatory element resulted in ectopic activity of the promoter in cortical cells of hypocotyls, roots and stems. A 169 bp fragment (–205 to –36) of the grp1.8 promoter conferred vascular-specific expression to CaMV 35S minimal promoters whereas a 141 bp fragment (–205 to –64) strongly activated these minimal promoters both in vascular and cortical cells. These experiments defined a new regulatory element (VSE) that is essential for vascular-specific expression and is located between –64 and –36. The 141 bp grp1.8 promoter sequence had enhancer-like properties as it was active in both orientations. A 24 bp sequence (bp –119 to –96, corresponding to the SE1 regulatory element) enhanced expression from several minimal promoters strongly but unspecifically, whereas a 26 bp sequence (–98 to –73, corresponding to the RSE regulatory element) induced vascular-specific expression. Thus, the grp1.8 promoter is regulated by a combinatorial mechanism that can integrate the action of different, non-additively acting regulatory elements into vascular-specific expression.     

Immunofluorescence of mineralocorticoid receptors in peripheral lymphocytes: Presence of receptor-like activity in patients with the autosomal dominant form of pseudohypoaldosteronism, and its absence in the recessive form

Pseudohypoaldosteronism is a syndrome characterized by salt wasting and a failure to thrive due to the resistance towards the action of aldosterone. Aldosterone levels and plasma renin activity are extremely elevated and aldosterone binding sites in peripheral mononuclear leukocytes have regularly shown to be reduced or absent. Sporadic as well as familial cases have been identified and an autosomal dominant as well as an autosomal recessive mode of inheritance has been described. A defect in the aldosterone receptor has been postulated, however, molecular genetic analysis in selected patients has not revealed a mutation in the sequence of the coding region of the cDNA of the mineralocorticoid receptor gene. In the present study we have used a fluorescence-labeled antibody to detect possible receptor expression in monocytes from patients with various clinical forms of pseudohypoaldosteronism. Patients with the sporadic as well as with the autosomal dominant form were clearly immunopositive despite being negative in terms of aldosterone receptor binding. In contrast in two patients with the autosomal recessive form there was no detectable receptor protein, consistent with the results obtained in the aldosterone binding studies. These results suggest that the pathogenesis of pseudohypoaldosteronism is heterogeneous not only regarding the mode of inheritance but also in terms of receptor binding. Thus, in a subgroup of patients the inability of the receptor to bind ligand may be due to a defect involving other, probably cellular factors rather than a deficiency or a defect in the mineralocorticoid receptor system itself.