Involvement of ferredoxin in desaturation of lipid-bound oleate in chloroplasts

Intact spinach (Spinacia oleracea) chloroplasts, pulse-labeled with [¹⁴C]acetate, desaturate newly formed fatty acids as ester groups of monogalactosyl diacylglycerol in a subsequent chase in the dark. Rupture of pulse-labeled chloroplasts by addition of a detergent solution 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate preserves part of this desaturation activity. Direct addition of different free fatty acids together with appropriate cofactors to detergent-ruptured chloroplasts results in fatty acid labeling of monogalactosyl diacylglycerol. During subsequent incubation these lipid-linked fatty acids are desaturated, i.e. 18:1 to 18:2 and 18:3 and to a small extent also 16:0 to 16:3. The formation of 18:2 was also observed after incorporation of 18:1 into sulfolipid and phosphatidyl choline. Density gradient centrifugation separated a membrane fraction from detergent-ruptured chloroplasts which in the presence of appropriate cofactors incorporated 18:1 and 18:2 into the above-mentioned lipids. In the light, desaturation was dependent on added ferredoxin, whereas in the dark, in addition to ferredoxin NAD(P)H was also required. Preliminary evidence for the involvement of membrane-bound ferredoxin:NADP oxidoreductase (FNR) as a third component of desaturation in the dark was obtained by inhibitor studies including antibodies against FNR. Desaturation of lipid-bound 18:1 and 18:2 resembles stearoyl-ACP desaturation with respect to its requirement of reduced ferredoxin and oxygen.     

Maltose excretion by the symbiotic Chlorella of the heliozoan Acanthocystis turfacea

Chlorella sp. strain 3.83, a symbiotic Chlorella isolated from the heliozoan Acanthocystis turfacea, excreted between 8% and 16% of assimilated ¹⁴CO₂ as maltose in the light (15000 lx), with a pH optimum around 4.8. This percentage increased when the illuminance was lowered (36% at 1700 lx). Release of [¹⁴C]maltose continued in darkness and could be inhibited by the uncoupler carbonyl cyanide p-trifluoro-methoxyphenylhydrazone and by diethylstilbestrol. Net efflux of maltose was observed even at a concentration ratio of extracellular/intracellular maltose of 7.8. Exogenous [¹⁴C]maltose (5 mM) was taken up by the cells with a rate <2% of that of simultaneous maltose release, indicating a practically unidirectional transport. It is concluded that maltose excretion is an active-transport process.Abbreviations DES diethylstilbestrol - FCCP carbonyl cyanide p-trifluoromethoxyphenyl hydrazone - p.c. packed cells This work was supported by the Deutsche Forschungsgemeinschaft. Thanks are due to Doris Meindl for skillful experimental help.     

A cyclic AMP response element mediates repression of tyrosine aminotransferase gene transcription by the tissue-specific extinguisher locus Tse-1

Tyrosine aminotransferase (TAT) gene expression is liver specific and inducible by glucocorticoids and via the cAMP signaling pathway. In fibroblasts and other nonliver cells the gene is subject to negative control by the trans-dominant tissue-specific extinguisher locus Tse-1. We identified a hepatocyte-specific enhancer that is repressed by Tse-1. Two distinct sequence motifs are absolutely essential for function of this enhancer: a cAMP response element (CRE), which is the target for repression by Tse-1, and a hepatocyte-specific element. The specificity of the enhancer is generated by the combination of these two essential elements, which are fully interdependent. In vivo footprinting indicates that Tse-1 acts by affecting protein binding at the CRE. A direct antagonism between Tse-1 and the cAMP signaling pathway suggests that Tse-1 plays a role in control of developmental activation of the TAT gene.     

The rate of bulk flow from the Golgi to the plasma membrane

A truncated analog of the backbone of sphingomyelin and glycolipids was synthesized. This truncated C8C8 ceramide was soluble in water (but was still able to cross cell membranes) and was utilized by the Golgi apparatus of living cells to produce water-soluble truncated phospholipids and glycolipids that were then secreted into the medium. Sphingomyelin is synthesized in a proximal (likely the cis) Golgi compartment. At 37 degrees C in CHO cells, the sphingomyelin analog is secreted with a half time of about 10 min. With this rate of bulk flow, no special signal is needed to pass through the Golgi to the plasma membrane. At 30 degrees C the half time of secretion of a lumenal ER marker is about 18 min, and that of the truncated sphingomyelin is about 14 min. Comparison of these rates sets an upper limit of about 4 min for half of the ER to be drained into the proximal Golgi at 30 degrees C.     

Tangential flow filtration and preliminary phylogenetic analysis of marine picoplankton

A procedure was developed for harvesting gram quantities of microbial biomass from oligotrophic waters, when mixed populations are present in low abundance. Picoplankton from Atlantic Ocean (Hydrostation S, Sargasso Sea) and Pacific Ocean (Aloha Station) sites were collected in a three-stage process: (i) collection of seawater through an intake covered with 10-microns-pore Nytex; (ii) concentration by a tangential flow filtration device equipped with 10 ft2 (0.929 m2) of 0.1-micron-pore fluorocarbon membrane; (iii) collection of cells from concentrate by centrifugation. The overall efficiency of picoplankton recovery was at least 37%. The cellular morphotypes recovered matched those of the original population. DNA was prepared from frozen cell pellets by enzymatic digestion, solvent extraction, and isopycnic centrifugation. As indicated by the binding of kingdom-specific hybridization probes to the purified DNA, the Sargasso Sea picoplankton in this collection were largely eubacteria.     

A microdeletion of less than 250 kb, including the proximal part of the FMR-1 gene and the fragile-X site, in a male with the clinical phenotype of fragile-X syndrome

A gene designated "FMR-1" has been isolated at the fragile-X locus. One exon of this gene is carried on a 5.1-kb EcoRI fragment that exhibits length variation in fragile-X patients because of amplification of or insertion into a CGG-repeat sequence. This repeat probably represents the fragile site. The EcoRI fragment also includes an HTF island that is hypermethylated in fragile-X patients showing absence of FMR-1 mRNA. In this paper, we present further evidence that the FMR-1 gene is involved in the clinical manifestation of the fragile-X syndrome and also in the expression of the cellular phenotype. A deletion including the HTF island and exons of the FMR-1 gene was detected in a fragile X-negative mentally retarded male who presented the clinical phenotype of the fragile-X syndrome. The deletion involves less than 250 kb of genomic DNA, including DXS548 and at least five exons of the FMR-1 gene. These data support the hypothesis that loss of function of the FMR-1 gene leads to the clinical phenotype of the fragile-X syndrome. In the fragile-X syndrome, there are pathogenetic mechanisms other than amplification of the CGG repeat that do have the same phenotypic consequences.     

Effect of magnesium on methanogenic subpopulations in a thermophilic acetate-degrading granular consortium.

The effects of Mg2+ on thermophilic (55 degrees C) granules grown on acetate in 0.2-liter upflow anaerobic sludge blanket reactors were studied. The methanogens in the granules were identified and counted by using antibody probes and the antigenic fingerprinting method. Packets of large coccoidal cells antigenically related to Methanosarcina thermophila TM-1 were scarce in the absence of Mg2+ but increased with increasing Mg2+ concentrations up to 30 mM; Methanosarcina packets immunologically related to Methanosarcina barkeri R1M3 showed a similar trend, and their numbers increased up to 100 mM Mg2+. The number of single cells antigenically related to TM-1, R1M3, and Methanosarcina mazei S-6 were scarce at low Mg2+ concentrations but increased drastically at 30 and 100 mM Mg2+. The number of rod-shaped bacteria antigenically related to Methanobacterium thermoautotrophicum GC1 and delta H was highest with no Mg2+ present, and their numbers decreased with increasing concentrations of the cation. These quantitative data, obtained by counting cells in suspensions made from disrupted granules, were confirmed by microscopic observation of the methanogenic subpopulations in thin histologic sections of the granules.     

In vitro reconstitution of a light-harvesting gene product: deletion mutagenesis and analyses of pigment binding.

AB96, a gene encoding a Pisum sativum chlorophyll a/b binding protein [Coruzzi et al. (1983) J. Biol. Chem. 258, 1399-1402], can be expressed in Escherichia coli and reconstituted with pigments by the procedure described by Plumley and Schmidt [(1987) Proc. Natl. Acad. Sci. U.S.A. 84, 146-150]. Following purification by polyacrylamide gel electrophoresis, the reconstituted pigment-protein complex (CP2) is shown to have similar pigment-binding characteristics to native CP2 complexes isolated from thylakoid membranes. Therefore, the AB96 gene product contains binding sites for chlorophylls a and b and xanthophylls, all of which are necessary for optimal reconstitution in vitro. Absorption, fluorescence, and circular dichroism spectroscopy indicate that the pigments are oriented accurately and that chlorophylls a and b are adjoined for energy transfer. Studies with proteins produced after deletion mutagenesis of AB96 indicate that NH2-terminal amino acids 1-21 and COOH-terminal amino acids 219-228 do not play a role in pigment binding. In contrast, amino acids 50-57 and 204-212 (encompassing one of three conserved histidine residues) are essential for reconstitution. Residues near the presumed NH2- and COOH-terminal alpha-helix boundaries (22-49 and 213-218, respectively) affect the stability of reconstituted CP2 during electrophoresis at 4 degrees C. Correlation of diminished chlorophyll a binding with disappearance of a negative circular dichroism near 684 nm suggests that amino acids 213-218 near the COOH-terminal boundary of the third membrane-spanning helix affect the binding of some chlorophyll a molecules.     

Aminoglycoside-3″-adenyltransferase confers resistance to spectinomycin and streptomycin in Nicotiana tabacum

The bacterial gene aad A encodes the enzyme aminoglycoside-3-adenyltransferase that confers resistance to spectinomycin and streptomycin in Escherichia coli. Chimeric genes have been constructed for expression in plants, and were introduced into Nicotiana tabacum by Agrobacterium binary transformation vectors. Spectinomycin or streptomycin in selective concentrations prevent greening of N. tabacum calli. Transgenic clones, however, formed green calli on selective media containing spectinomycin, streptomycin, or both drugs. Resistance was inherited as a dominant Mendelian trait in the seed progeny. Resistance conferred by the chimeric aad A gene can be used as a color marker similar to the resistance conferred by the streptomycin phosphotransferase gene to streptomycin.