Primary structure of a photoactive yellow protein from the phototrophic bacterium Ectothiorhodospira halophila,with evidence for the mass and the binding site of the chromophore.

The complete amino acid sequence of the 125-residue photoactive yellow protein (PYP) from Ectothiorhodospira halophila has been determined to be MEHVAFGSEDIENTLAKMDDGQLDGLAFGAIQLDGDGNILQYNAAEGDITGRDPKEVIGKNFFKDVAP+ ++ CTDSPEFYGKFKEGVASGNLNTMFEYTFDYQMTPTKVKVHMKKALSGDSYWVFVKRV. This is the first sequence to be reported for this class of proteins. There is no obvious sequence homology to any other protein, although the crystal structure, known at 2.4 A resolution (McRee, D.E., et al., 1989, Proc. Natl. Acad. Sci. USA 86, 6533-6537), indicates a relationship to the similarly sized fatty acid binding protein (FABP), a representative of a family of eukaryotic proteins that bind hydrophobic molecules. The amino acid sequence exhibits no greater similarity between PYP and FABP than for proteins chosen at random (8%). The photoactive yellow protein contains an unidentified chromophore that is bleached by light but recovers within a second. Here we demonstrate that the chromophore is bound covalently to Cys 69 instead of Lys 111 as deduced from the crystal structure analysis. The partially exposed side chains of Tyr 76, 94, and 118, plus Trp 119 appear to be arranged in a cluster and probably become more exposed due to a conformational change of the protein resulting from light-induced chromophore bleaching. The charged residues are not uniformly distributed on the protein surface but are arranged in positive and negative clusters on opposite sides of the protein. The exact chemical nature of the chromophore remains undetermined, but we here propose a possible structure based on precise mass analysis of a chromophore-binding peptide by electrospray ionization mass spectrometry and on the fact that the chromophore can be cleaved off the apoprotein upon reduction with a thiol reagent. The molecular mass of the chromophore, including an SH group, is 147.6 Da (+/- 0.5 Da); the cysteine residue to which it is bound is at sequence position 69.     

Ornibactins—a new family of siderophores from Pseudomonas

Novel linear hydroxamate/hydroxycarboxylate siderophores from strains of Pseudomonas cepacia were isolated and named ornibactins. The ornibactins represent modified tetrapeptide siderophores, possessing the sequence l-Orn¹(N -OH, N -acyl)-d-threo-Asp(-OH)-l-Ser-l-Orn⁴(N -OH, N -formyl)-1,4-diaminobutane. The N -acyl groups of Orn¹(N -OH, N -acyl) may vary and represent the three acids 3-hydroxybutanoic acid, 3-hydroxyhexanoic acid and 3-hydroxyoctanoic acid, leading to a mixture of three different ornibactins, designated according to their acyl chain length as ornibactin-C4, ornibactin-C6 and ornibactin-C8. Each of the siderophores is accompanied by a small amount of a more hydrophilic component with a 16 a.m.u. higher mass. The structure elucidation was based on results from gas chromatography amino acid analysis, electrospray mass spectrometry, and one- and two-dimensional nuclear magnetic resonance techniques.     

Growth-related gene expression in Nicotiana tabacum mesophyll protoplasts

Eight cDNAs whose genes are more strongly expressed in suspension cells in growth phase than in stationary phase and at a low level in mature leaves have been isolated. The corresponding mRNAs are abundantly accumulated in young plant organs and in germinating seeds but are almost undetectable in mature plant tissues and dry seeds. Six of these cDNAs were characterized by comparison of nucleotide and protein sequences to the EMBL and SWISSPROT databanks. These eight growth-related genes are expressed in protoplasts isolated from Nicotiana tabacum mesophyll cells shortly after preparation (4 h). Two of them are expressed in freshly isolated protoplasts (early genes), while the other six are detected after 4 h of culture (late genes). Seven are more abundantly expressed in protoplasts than in growing plant organs while one growth-related gene is weakly expressed in protoplasts, as is the histone H₄ gene. They seem to be induced in protoplasts by a synergistic effect of wounding and maceration. Sustained expression of the early genes is dependent on the presence of sucrose in the culture medium.     

First chromosome scale genomes of ithomiine butterflies (Nymphalidae: Ithomiini): Comparative models for mimicry genetic studies

The ithomiine butterflies (Nymphalidae: Danainae) represent the largest known radiation of Müllerian mimetic butterflies. They dominate by number the mimetic butterfly communities, which include species such as the iconic neotropical Heliconius genus. Recent studies on the ecology and genetics of speciation in Ithomiini have suggested that sexual pheromones, colour pattern and perhaps hostplant could drive reproductive isolation. However, no reference genome was available for Ithomiini, which has hindered further exploration on the genetic architecture of these candidate traits, and more generally on the genomic patterns of divergence. Here, we generated high-quality, chromosome-scale genome assemblies for two Melinaea species, M. marsaeus and M. menophilus, and a draft genome of the species Ithomia salapia. We obtained genomes with a size ranging from 396 to 503 Mb across the three species and scaffold N50 of 40.5 and 23.2 Mb for the two chromosome-scale assemblies. Using collinearity analyses we identified massive rearrangements between the two closely related Melinaea species. An annotation of transposable elements and gene content was performed, as well as a specialist annotation to target chemosensory genes, which is crucial for host plant detection and mate recognition in mimetic species. A comparative genomic approach revealed independent gene expansions in ithomiines and particularly in gustatory receptor genes. These first three genomes of ithomiine mimetic butterflies constitute a valuable addition and a welcome comparison to existing biological models such as Heliconius, and will enable further understanding of the mechanisms of adaptation in butterflies.     

Biochemical bone marrow markers and their significance in postmenopausal osteoporosis--a new method in the diagnosis of osteoporosis?]

This study analyzes the qualification of biochemical markers in the diagnosis of osteoporosis and evaluates the potential of a multiparametric classification of premenopausal and non-osteoporotic as well as osteoporotic postmenopausal women, which is based on biochemical marker profiles. For this evaluation data of 29 women in the age between 28-74 years were used. The classification of osteoporosis was done by the trabecular density of the lumbar spine using qCT-measurements. The biochemical markers of formation and resorption AP, bAP, OC, ucOC, PICP, PYD, DPD, NTX, BSP and vitamin K were analyzed on day 1 and 42 in all patients. For vitamin K we found significant distribution differences between non-osteoporotic and osteoporotic women (p<0.005). The crosslinks PYD and DPD showed weakly significant differences. All other parameters exhibited non-significant results. Vitamin K acted with a sensitivity of 64% and a specificity of 82%. The used multiparameter classification process improved sensitivity and specificity considerably. The parameter profiles of OC/PYD, vitamin K/PYD and vitamin K/bAP revealed the highest sensitivities with specificities of more than 82%.     

Distribution of Plasma Membrane H+-ATPase and Polar Current Patterns in Leaves and Stems of Elodea canadensis*

The proton pumping ATPase in the plasma membrane of Elodea canadensis is believed to play a major role in inorganic carbon acquisition. To investigate potentially different carbon uptake strategies within the same plant, plasma membrane H⁺-ATPase distribution and polar current patterns were investigated in Elodea leaves and stems. Specific activity of plasma membrane H⁺-ATPase in leaf microsomal fractions was tenfold higher than in stem derived microsomes. Probing western blots with a monoclonal antibody specific for plasma membrane H⁺-ATPase, yielded strongly visible double bands at 100 kDa in leaf microsome preparations, whereas little antigen was detected in analogous stem microsome preparations. Using the same plasma membrane H⁺-ATPase specific antibody on tissue sections, the enzyme was found almost exclusively localized at the border of cells at the lower leaf surface. A positive ion current leaving the lower leaf surface was measured, using a vibrating probe device. Part of this current entered the upper leaf surface and part of it the internodes of the stem. The experimental results support the view, that Elodea leaves have different means of inorganic carbon uptake than stem internodes.     

Cloning and characterization of theNeisseria gonorrhoeae MS11folC gene

The gene coding for folylpoly-(γ)-glutamate synthetase (FPGS)-dihydrofolate synthetase (DHFS) ofNeisseria gonorrhoeae (Ngo) has been cloned by functional complementation of anEscherichia coli folC mutant (SF4). The sequence encodes a 224-residue protein of 46.4 kDa. It shows 46% identity to theE. coli FPGS-DHFS and 29% identity to the PFGS ofLactobacillus casei. Sequence comparisons between the three genes reveal regions of high homology, including ATP binding sites required for bifunctionality, all of which may be important for FPGS activity. In contrast toL. casei FPGS, theE. coli andNgo enzymes share some additional regions which may be essential for DHFS activity. The products ofNgo folC and flanking genes were monitored by T7 promoter expression. Interestingly, deletion of the upstreamfolI gene, which encodes a 16.5 kDa protein, abolishes the capacity offolC to complementE. coli SF4 to the wild-type phenotype. The ability to complement can be restored byfolI providedin trans. UnlikefolC mutants, gonococcalfolI mutants are viable and display no apparent phenotype. Thus, in contrast toE. coli, Ngo folC is expressed at a sufficiently high level from its own promoter, in the absence of FolI. This study provides the first insights into the genetic complexity of one-carbon metabolism inNgo.     

Exploiting the complete yeast genome sequence

The completion of the genome sequence of the budding yeast Saccharomyces cerevisiae marks the dawn of an exciting new era in eukaryotic biology that will bring with it a new understanding of yeast, other model organisms, and human beings. This body of sequence data benefits yeast researchers by obviating the need for piecemeal sequencing of genes, and allows researchers working with other organisms to tap into experimental advantages inherent in the yeast system and learn from functionally characterized yeast gene products which are their proteins of interest. In addition, the yeast post-genome sequence era is serving as a testing ground for powerful new technologies, and proven experimental approaches are being applied for the first time in a comprehensive fashion on a complete eukaryotic gene repertoire.     

Mutational studies of conserved residues in the dimer interface of nerve growth factor.

An understanding of the structure-function relationship of nerve growth factor (NGF) requires precise knowledge of all the residues and regions that participate in NGF receptor binding, receptor activation, and biological activity. Seven recombinant human NGF mutants having alanine substituted for residues located either in the NGF dimer interface or beta-strand region were studied to determine the role of each amino acid residue in NGF biological activity. F86A, T91A, R100A, and R103A remained nearly full active with 61, 120, 91, and 73% of wild-type activity, respectively, in the PC12 cell bioassay. Hydrophobic core and dimer interface residues Y52, F53, and F54 were studied in more detail. Y52A and F54A were expressed in very low levels, suggesting that these two residues may be important for protein stability. Y52A retained full biological activity (91%). F53A had a 20- and 70-fold reduction in biological activity and TrkA phosphorylation, respectively, with only a 5- to 10-fold effect on TrkA binding and no effect on low-affinity receptor binding. F54A had significantly decreased TrkA phosphorylation and biological activity (40-fold). The results suggest that F53 and F54 may play a structural role in TrkA receptor activation subsequent to binding.