Evolutionary relationships among aminotransferases. Tyrosine aminotransferase, histidinol-phosphate aminotransferase, and aspartate aminotransferase are homologous proteins

A data base was compiled containing the amino acid sequences of 12 aspartate aminotransferases and 11 other aminotransferases. A comparison of these sequences by a standard alignment method confirmed the previously reported homology of all aspartate aminotransferases and Escherichia coli tyrosine aminotransferase. However, no significant similarity between these proteins and any of the other aminotransferases was detected. A more rigorous analysis, focusing on short sequence segments rather than the total polypeptide chain, revealed that rat tyrosine aminotransferase and Saccharomyces cerevisiae and Escherichia coli histidinol-phosphate aminotransferase share several homologous sequence segments with aspartate aminotransferases. For comparison of the complete sequences, a multiple sequence editor was developed to display the whole set of amino acid sequences in parallel on a single work-sheet. The editor allows gaps in individual sequences or a set of sequences to be introduced and thus facilitates their parallel analysis and alignment. Several clusters of invariant residues at corresponding positions in the amino acid sequences became evident, clearly establishing that the cytosolic and the mitochondrial isoenzyme of vertebrate aspartate aminotransferase, E. coli aspartate aminotransferase, rat and E. coli tyrosine aminotransferase, and S. cerevisiae and E. coli histidinol-phosphate aminotransferase are homologous proteins. Only 12 amino acid residues out of a total of about 400 proved to be invariant in all sequences compared; they are either involved in the binding of pyridoxal 5'-phosphate and the substrate, or appear to be essential for the conformation of the enzymes.     

Molecular characterization of the virulence gene virA of the Agrobacterium tumefaciens octopine Ti plasmid

The virulence loci play an essential role in tumor formation by Agrobacterium tumefaciens. Induction of vir gene expression by plant signal molecules is solely dependent on the virulence loci virA and virG. This study focused on the virA locus of the octopine type Ti plasmid pTi15955. The nucleic acid sequence of a 5.7-kilobase fragment encompassing virA was determined. Genetic analysis of this region revealed that virA contains one open reading frame coding for a protein of 91 639 daltons. Immunodetection with antibodies raised against a 35-kDa VirA fusion protein produced in E. coli identified the VirA product in wild-type Agrobacterium cells. Moreover, it is shown that the VirA protein is located in the cytoplasmic membrane fraction of Agrobacterium. These data confirm the proposed regulatory function of VirA whereby VirA acts as a membrane sensor protein to identify plant signal molecules in the environment. The proposed sensory function of VirA strikingly resembles the function of the chemotaxis receptor proteins of E. coli.     

Pigment organization and energy transfer in the green photosynthetic bacterium Chloroflexus aurantiacus. III. Energy transfer in whole cells

The transfer of excitation energy in intact cells of the thermophilic green photosynthetic bacterium Chloroflexus aurantiacus was studied both at low temperature and under more physiological conditions. Analysis of excitation spectra measured at 4K indicates that the minor fraction of bacteriochlorophyll a present in the chlorosome functions as an intermediate in energy transfer between the main light-harvesting pigment BChl c and the membrane-bound B808-866 antenna complex. This supports the hypothesis that BChl a is associated with the base plate which connects the chlorosome with the membrane. The overall efficiency for energy transfer from the chlorosome to the membrane is only 15% at 4K. High efficiencies of close to 100% are observed above 40°C near the temperature where the cultures are grown. Cooling to 20°C resulted in a sudden drop of the transfer efficiency which appeared to originate in the chlorosome. This decrease may be related to a lipid phase transition. Further cooling mainly affected the efficiency of transfer between the chlorosome and the membrane. This effect can only partially be explained by a decreased Förster overlap between the chlorosomal BChl a and BChl a 808 associated with the membrane-bound antenna system. The temperature dependence of the fluorescence yield of BChl a 866 also appeared to be affected by lipid phase transitions, suggesting that this fluorescence can be used as a native probe of the physical state of the membrane.     

Mass-spectrometric quantitation of cytokinins in tobacco crown-gall tumours induced by mutated octopine Ti plasmids of Agrobacterium tumefaciens

The levels of the major cytokinins, zeatin, zeatin riboside, zeatin riboside-5-monophosphate and zeatin-7-glucoside were measured in tobacco (Nicotiana tabacum L.) crown-gall tissues carrying insertion and deletion mutations in the T-DNA. Measurements were made by combined gas chromatography-mass spectrometry using selected ion monitoring with ¹⁵N- and ²H-labelled internal standards. The results demonstrate that, relative to wild-type tumour tissue, cytokinin levels are considerably elevated in tissues lacking functional T-DNA auxin-biosynthetic genes. From a detailed analysis of the major cytokinin metabolites it is concluded that a reduction in the extent of cytokinin degradation via N⁶-side-chain cleavage is an important factor leading to increased cytokinin levels in these tissues.Abbreviations IAA indole-3-acetic acid - SIM selected ion monitoring - Z zeatin - [7G]Z zeatin-7-glucoside - [9R]Z zeatin-9-riboside - [9R-5P]Z zeatin riboside-5-monophosphate     

Interaction of lymphocytes and high endothelial venules in irradiated lymph nodes

After total-body exposure to various doses of ionizing radiation, the ability of lymphocytes to interact specifically with high endothelial venules of rat cervical and mesenteric lymph nodes was analyzed in frozen sections. Following a radiation dose of 1.5 Gy, high endothelial venules remained intact and the binding of unirradiated lymphocytes to the venules was enhanced relative to unirradiated controls. At radiation doses above 5.0 Gy, damage to high endothelial venules was observed histologically as well as assessed functionally. There was a significant decrease in specific lymphocyte-venule binding and a significant increase in nonspecific binding. These findings suggest that radiation-induced damage to high endothelial venules might play a role in radiation-induced immunosuppression by interfering with the normal passage of lymphocytes from the blood into lymph nodes via a specific interaction between lymphocytes and high endothelial venules.     

Genetic basis of virulence in Shigella species.

Shigella species and enteroinvasive strains of Escherichia coli cause disease by invasion of the colonic epithelium, and this invasive phenotype is mediated by genes carried on 180- to 240-kb plasmids. In addition, at least eight loci on the Shigella chromosome are necessary for full expression of virulence. The products of these genes can be classified as (i) virulence determinants that directly affect the ability of shigellae to survive in the intestinal tissues, e.g., the aerobactin siderophore (iucABCD and iutA), superoxide dismutase (sodB), and somatic antigen expression (rfa and rfb); (ii) cytotoxins that contribute to the severity of disease, e.g., the Shiga toxin (stx) and a putative analog of this toxin (flu); and (iii) regulatory loci that affect the expression of plasmid genes, e.g., ompR-envZ, which mediates response to changes in osmolarity, virR (osmZ), which mediates response to changes in temperature, and kcpA, which affects the translation of the plasmid virG (icsA) gene which is associated with intracellular bacterial mobility and intracellular bacterial spread. A single plasmid regulatory gene (virF) controls a virulence-associated plasmid regulon including virG (icsA) and two invasion-related loci, i.e., (i) ipaABCD, encoding invasion plasmid antigens that may be structural components of the Shigella invasion determinant; and (ii) invAKJH (mxi), which is necessary for insertion of invasion plasmid antigens into the outer membrane.     

The root-knot nematode resistance gene (Mi) in tomato: construction of a molecular linkage map and identification of dominant cDNA markers in resistant genotypes

A dominant allele at the Mi locus on chromosome 6 of tomato (Lycopersicon esculentum Mill) confers resistance to three species of root-knot nematodes (Meloidogyne). The resistance, which is associated with a localized necrotic response, was originally introduced into tomato from the wild species Lycopersicon peruvianum. As a step towards the molecular cloning of Mi, we have identified closely linked DNA markers from both cDNA and genomic DNA libraries as restriction fragment length polymorphisms (RFLPs). DNA from tomato populations segregating for nematode resistance was analyzed to generate a high-resolution genetic map of this region. Additional information on gene order was obtained by comparing the size of the introgressed L. peruvianum chromosomal segment within a collection of nematode-resistant tomato lines. Among the four cDNA markers that are tightly linked to Mi, three are dominant, i.e. L. peruvianum-specific. One cDNA marker corresponds to a gene family comprising 20-30 members, one of which is diagnostic for all nematode-resistant genotypes tested. The presence of non-homologous sequences around the Mi gene may contribute to the suppression of recombination in this region of the genome in crosses heterozygous for Mi. The potential of 'walking' from closely linked markers to Mi is discussed.     

Experimental effects of elevated salinity on three benthic invertebrates in Pyramid Lake,Nevada

Salinity of Pyramid Lake increased from 3.7 to 5.5 between 1933 and 1980. Concern over future reductions in overall species richness prompted experiments to assess responses of dominant lake organisms to elevated salinity. Salinity tolerances of three important benthic invertebrates, Hyalella aztecta, Chironomus utahensis, and Heterocypris sp., were tested in controlled laboratory bioassays and also in a semi-natural environment consisting of large (47 m³) mesocosms.Densities of H. azteca in mesocosms were significantly lower at salinities of 8.0 and 11.0 compared with 5.6 controls in year one, but not in 8.5 salinity mesocosms in year two. The 96-h LC₅₀ for H. azteca was high at 19.5. Short-term mortalities of C. utahensis were 100% at salinities of 13.3 and greater. Fifty-seven percent fewer larvae matured from third to fourth instar at 8.9 than at 5.5 salinity in 17 day subacute bioassays. Furthermore, larval chironomid densities and emergence of adults from mesocosms were significantly reduced at salinities of 8.0 and higher compared with controls. Mortality of Heterocypris sp. was 50% at a salinity of 18.6 in laboratory bioassays and populations in mesocosms ranged between 40 and 100% lower at salinities of 8.0 and 11.0 than in controls.Multiple generation mesocosm experiments indicated all three invertebrates were more sensitive to elevated salinity than results of short-term bioassays. Our studies suggest populations of these invertebrates may be reduced from present levels if Pyramid Lake's salinity were to double, although none are expected to be extirpated. Food habit shifts and reduced production of lake fishes are likely consequences of salinity-induced disruption in the benthic invertebrate forage base.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)