Molecular analysis of theArabidopsis pattern formation geneGNOM: gene structure and intragenic complementation

TheGNOM gene is required for pattern formation along the main body axis of the embryo in the flowering plantArabidopsis thaliana. Mutations in theGNOM gene alter the asymmetric division of the zygote and interfere with the formation of distinct apical-basal regions in the developing embryo. We have isolated theGNOM gene by positional cloning, characterised its structure and determined the molecular lesions in mutant alleles. Although the predicted 163 kDa GNOM protein has a conserved domain in common with the yeast secretory protein Sec7p, it is most closely related in size and overall similarity to the product of the yeastYEC2 gene, which is not essential for cell viability. Four fully complementinggnom alleles carry missense mutations in conserved regions, seven partially complementing alleles have premature stop codon mutations and two non-complementing alleles have splice-site lesions. Our results suggest that the GNOM protein acts as a complex of identical subunits and that partial complementation may involve low levels of full-length protein generated by inefficient translational read-through.Communicated by H. Saedler     

Formation of intracytoplasmic lipid inclusions by Rhodococcus opacus strain PD630

An oleaginous hydrocarbon-degrading Rhodococcus opacus strain (PD630) was isolated from a soil sample. The cells were able to grow on a variety of substrates and to produce large amounts of three different types of intracellular inclusions during growth on alkanes, phenylalkanes, or non-hydrocarbon substrates. Electron microscopy revealed large numbers of electron-transparent inclusions with a sphere-like structure. In addition, electron-dense inclusions representing polyphosphate and electron-transparent inclusions with an elongated disc-shaped morphology occurred in small amounts. The electron-transparent inclusions of alkane- or gluconate-grown cells were composed of neutral lipids (98%, w/w), phospholipids (1.2%, w/w), and protein (0.8%, w/w). The major component of the cellular inclusions was triacylglycerols; minor amounts of diacylglycerols and probably also some free fatty acids were also present. Free fatty acids and/or fatty acids in acylglycerols in cells of R. opacus amounted up to 76 or 87% of the cellular dry weight in gluconate- or olive-oil-grown cells, respectively. The fatty acid composition of the inclusions depended on the substrate used for cultivation. In cells cultivated on n-alkanes, the composition of the fatty acids was related to the substrate, and intermediates of the β-oxidation pathway, such as hexadecanoic or pentadecanoic acid, were among the acylglycerols. Hexadecanoic acid was also the major fatty acid (up 36% of total fatty acids) occurring in the lipid inclusions of gluconate-grown cells. This indicated that strain PD630 utilized β-oxidation and de novo fatty acid biosynthesis for the synthesis of storage lipids. Inclusions isolated from phenyldecane-grown cells contained mainly the non-modified substrate and phenylalkanoic acids derived from the hydrocarbon oxidation, such as phenyldecanoic acid, phenyloctanoic acid, and phenylhexanoic acid, and approximately 5% (w/w) of diacylglycerols. The lipid inclusions seemed to have definite structures, probably with membranes at their surfaces, which allow them to maintain their shape, and with some associated proteins, probably involved in the inclusion formation. Received: 22 December 1995 / Accepted: 12 March 1996     

Nitric oxide synthase in the rat carotid body and carotid sinus

The participation of nitric oxide synthase (NOS) in the innervation of the rat carotid body and carotid sinus was investigated by means of NADPH-diaphorase histochemistry and NOS immunohistochemistry using antisera raised against purified neuronal NOS and a synthetic tridecapeptide. NOS was detected in 23% of neurons at the periphery of the carotid bodies. Some negative neurons were surrounded by NOS-positive terminals. NOS-containing varicose nerve fibres innervated the arterial vascular bed and, to a lesser extent, the islands of glomus cells. These fibres persisted after transection of the carotid sinus nerve and are probably derived from intrinsic neurons. Large NOS-positive axonal swellings in the wall of the carotid sinus were absent after transection of the sinus nerve, indicating their sensory origin. The results suggest a neuronal nitrergic control of blood flow, neuronal activity and chemoreception in the carotid body, and an intrinsic role of NO in the process of arterial baroreception.     

Multiplex PCR for detection of the heat-labile toxin gene and shiga-like toxin I and II genes in Escherichia coli isolated from natural waters.

A triplex PCR method was developed to simultaneously amplify a heat-labile toxin sequence (LT) of 258 bp, a shiga-like toxin I sequence (SLT I) of 130 bp, and a shiga-like toxin II sequence (SLT II) of 346 bp from toxigenic strains of Escherichia coli. This method was used to screen 377 environmental E. coli isolates from marine waters or estuaries located in Southern California and North Carolina for enterotoxigenic or enterohemorrhagic E. coli strains. Of the 377 E. coli screened, one isolate was found to belong to the enterotoxigenic group, since it contained a LT homologous sequence, and one isolate was found to belong to the enterohemorrhagic group, since it contained a SLT I homologous sequence. None was found to contain SLT II homologous sequences. The pathogenicity of the positive environmental E. coli isolates was confirmed by standard bioassays with Y-1 adrenal cells and Vero cells to confirm toxin production. Our results suggest that toxigenic E. coli occurs infrequently in environmental waters and that there is a low public health risk from toxigenic E. coli in coastal waters.     

Autogenous translational regulation of the ribosomal MvaL1 operon in the archaebacterium Methanococcus vannielii.

The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaL1, MvaL10, and MvaL12, encoded by the MvaL1 operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaL1, the homolog of the regulatory protein L1 encoded by the L11 operon of Escherichia coli, was shown to be an autoregulator of the MvaL1 operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaL1 was localized by site-directed mutagenesis to a region within the coding sequence of the MvaL1 gene commencing about 30 bases downstream of the ATG initiation codon. The MvaL1 binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the L1 regulatory target site of E. coli and to the putative binding site for MvaL1 on the 23S rRNA. In contrast to other regulatory systems, the putative MvaL1 binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.     

FTIR spectroscopic study of the dynamics of conformational substates in hydrated carbonyl-myoglobin films via temperature dependence of the CO stretching band parameters.

Two hydrated carbonyl myoglobin (MbCO) films, one containing (0.30 g water)/(g MbCO) from MbCO solution in water at pH 5.5 and the other (0.32 g water)/(gMbCO) from 0.1 M potassium phosphate buffer solution at pH 6.8, were studied by FTIR spectroscopy from 293 K to 78 K at selected temperatures on cooling and reheating. Above approximately 180 K the general trend in temperature dependence of half-bandwidths, peak maxima, and band area ratios of the A1 and A3 conformer bands is similar to those reported by Ansari et al. (1987. Biophys. J. 26:337) for MbCO in 75% glycerol/water solution, but abrupt changes in slopes at approximately 180-200 K and freezing-in of conformer populations, which could be taken as indicator for glass transition of the solvent or the protein, are absent for the hydrated MbCO films. This is interpreted in terms of an exceptionally broad distribution of relaxation times, and is in accord with conclusions from recent calorimetric annealing studies of hydrated protein powders (Sartor et al. 1994. Biophys. J. 66:249). Exchange between the three A conformers does not stop at approximately 180-200 K but occurs over the whole temperature region studied. These results are then discussed with respect to MbCO's behavior in the glass-->liquid transition region of glass-forming solvents, and it is concluded that, in analogy to the behavior of low-molecular-weight compounds with a distribution of rapidly interconverting conformers, freezing-in of MbCO's A conformer populations by the solvent should not be mistaken for a glass transition of MbCO.     

Chloroplast DNA phylogeny of Lens (Leguminosae): origin and diversity of the cultivated lentil

A restriction-site analysis of chloroplast DNA (cpDNA) variation in Lens was conducted to: (1) assess the levels of variation in Lens culinaris ssp. culinaris (the domesticated lentil), (2) identify the wild progenitor of the domesticated lentil, and (3) construct a cpDNA phylogeny of the genus. We analyzed 399 restriction sites in 114 cultivated accessions and 11 wild accessions. All but three accessions of the cultivar had identical cpDNAs. Two accessions exhibited a single shared restriction-site loss, and a small insertion was observed in the cpDNA of a third accession. We detected 19 restriction-site mutations and two length mutations among accessions of the wild taxa. Three of the four accessions of L. culinaris ssp. orientalis were identical to the cultivars at every restriction site, clearly identifying ssp. orientalis as the progenitor of the cultivated lentil. Because of its limited cpDNA diversity, we conclude that either the cultivated lentil has passed through a genetic bottleneck during domestication and lost most of its cytoplasmic variability or else was domesticated from an ancestor that was naturally depauperate in cpDNA restriction-site variation. However, because we had access to only a small number of populations of the wild taxa, the levels of variation present in ssp. orientalis can only be estimated, and the extent of such a domestication bottleneck, if applicable, cannot be evaluated. The cpDNA-based phylogeny portrays Lens as quite distinct from its putative closest relative, Vicia montbretii. L. culinaris ssp. odemensis is the sister of L. nigricans; L. culinaris is therefore paraphyletic given the current taxonomic placement of ssp. odemensis. Lens nigricans ssp. nigricans is by far the most divergent taxon of the genus, exhibiting ten autapomorphic restriction-site mutations.     

Neuronal and endothelial nitric oxide synthase immunoreactivity and NADPH-diaphorase staining in rat and human pancreas: influence of fixation

In this study, we wished to clarify the distribution and co-localization of nitric oxide synthase and NADPH-diaphorase (NADPH-d) in nerve cells, nerve fibres and parenchymal cells in exocrine and endocrine pancreas, and to assess the influence of fixation on the staining pattern obtained. For this purpose, we applied nitric oxide synthase immunocytochemistry and NADPH-d histochemistry to rat and human pancreas under different fixation conditions. Antibodies to neuronal and endothelial nitric oxide synthase were similarly applied. We found complete co-localization of neuronal nitric oxide synthase and NADPH-d in ganglion cells, and in nerve fibres around acini, excretory ducts, blood vessels and in islets of Langerhans of rat and human pancreas. Immunoreactivity for endothelial nitric oxide synthase was co-localized with NADPH-d in endothelial cells. However, in NADPH-d reactive islet and ductal epithelial cells we could detect neither brain nor endothelial nitric oxide synthase immunoreactivity with any fixation protocol applied. There were marked differences in NADPH-d staining of both neurons and parenchymal cells under different fixation conditions. These results indicate the existence of different types of NADPH-d, which are associated or not associated with nitric oxide synthase(s), and which are differently influenced by various fixation procedures in rat and human pancreas.     

Ammonium rhythm in cultures of the cyanobacterium Microcystis firma

Over a period of several days, rhythmic changes in extracellular NH⁺₄ concentration take place in cultures of the cyanobacterium Microcystis firma (Bré et Lenorm.) Schmidle, strain Gromov/St. Petersb. 398, under conditions of restricted CO₂ supply and light/dark alternation. The changes are enhanced by nitrate supply. Among the various processes generating intracellular NH⁺₄ (NH⁴₄ uptake, NO⁻₃ reduction, protein and amino acid degradation, photorespiration), NO⁻₃ reduction appears as the one most important. This can be concluded from experiments with and without nitrate and/or ammonium in the medium. In the presence of saturating CO₂, continuous light, or continuous darkness, rhythmic NH⁺⁴₄ oscillations are not induced. Studies of the incorporation of NH⁺₄ nitrogen by in vivo ¹⁵N-NMR show that if CO₂ is supplied, ¹⁵N is accumulated in several components with the following time course: in the first hour in Gln (δ), in the second hour in the α-amino groups of most nonbranched amino acids, in the third hour in γ-aminobutyric acid (GABA), Orn (δ) and Lys (ε), and in the sixth hour in Ala. Carbon limitation, however, results in accumulation of label in the amide nitrogen of glutamine only.