Mice have a transcribed L-threonine aldolase/GLY1 gene, but the human GLY1 gene is a non-processed pseudogene

BACKGROUND: There are three pathways of L-threonine catabolism. The enzyme L-threonine aldolase (TA) has been shown to catalyse the conversion of L-threonine to yield glycine and acetaldehyde in bacteria, fungi and plants. Low levels of TA enzymatic activity have been found in vertebrates. It has been suggested that any detectable activity is due to serine hydroxymethyltransferase and that mammals lack a genuine threonine aldolase. RESULTS: The 7-exon murine L-threonine aldolase gene (GLY1) is located on chromosome 11, spanning 5.6 kb. The cDNA encodes a 400-residue protein. The protein has 81% similarity with the bacterium Thermotoga maritima TA. Almost all known functional residues are conserved between the two proteins including Lys242 that forms a Schiff-base with the cofactor, pyridoxal-5'-phosphate. The human TA gene is located at 17q25. It contains two single nucleotide deletions, in exons 4 and 7, which cause frame-shifts and a premature in-frame stop codon towards the carboxy-terminal. Expression of human TA mRNA was undetectable by RT-PCR. In mice, TA mRNA was found at low levels in a range of adult tissues, being highest in prostate, heart and liver. In contrast, serine/threonine dehydratase, another enzyme that catabolises L-threonine, is expressed very highly only in the liver. Serine dehydratase-like 1, also was most abundant in the liver. In whole mouse embryos TA mRNA expression was low prior to E-15 increasing more than four-fold by E-17. CONCLUSION: Mice, the western-clawed frog and the zebrafish have transcribed threonine aldolase/GLY1 genes, but the human homolog is a non-transcribed pseudogene. Serine dehydratase-like 1 is a putative L-threonine catabolising enzyme

The molecular phylogeny of eph receptors and ephrin ligands

<p>Abstract</p> <p>Background</p> <p>The tissue distributions and functions of Eph receptors and their ephrin ligands have been well studied, however less is known about their evolutionary history. We have undertaken a phylogenetic analysis of Eph receptors and ephrins from a number of invertebrate and vertebrate species.</p> <p>Results</p> <p>Our findings indicate that Eph receptors form three major clades: one comprised of non-chordate and cephalochordate Eph receptors, a second comprised of urochordate Eph receptors, and a third comprised of vertebrate Eph receptors. Ephrins, on the other hand, fall into either a clade made up of the non-chordate and cephalochordate ephrins plus the urochordate and vertebrate ephrin-Bs or a clade made up of the urochordate and vertebrate ephrin-As.</p> <p>Conclusion</p> <p>We have concluded that Eph receptors and ephrins diverged into A and B-types at different points in their evolutionary history, such that primitive chordates likely possessed an ancestral ephrin-A and an ancestral ephrin-B, but only a single Eph receptor. Furthermore, ephrin-As appear to have arisen in the common ancestor of urochordates and vertebrates, whereas ephrin-Bs have a more ancient bilaterian origin. Ancestral ephrin-B-like ligands had transmembrane domains; as GPI anchors appear to have arisen or been lost at least 3 times.</p

EphA3 Expressed in the Chicken Tectum Stimulates Nasal Retinal Ganglion Cell Axon Growth and Is Required for Retinotectal Topographic Map Formation

BACKGROUND: Retinotopic projection onto the tectum/colliculus constitutes the most studied model of topographic mapping and Eph receptors and their ligands, the ephrins, are the best characterized molecular system involved in this process. Ephrin-As, expressed in an increasing rostro-caudal gradient in the tectum/colliculus, repel temporal retinal ganglion cell (RGC) axons from the caudal tectum and inhibit their branching posterior to their termination zones. However, there are conflicting data regarding the nature of the second force that guides nasal axons to invade and branch only in the caudal tectum/colliculus. The predominant model postulates that this second force is produced by a decreasing rostro-caudal gradient of EphA7 which repels nasal optic fibers and prevents their branching in the rostral tectum/colliculus. However, as optic fibers invade the tectum/colliculus growing throughout this gradient, this model cannot explain how the axons grow throughout this repellent molecule. METHODOLOGY/PRINCIPAL FINDINGS: By using chicken retinal cultures we showed that EphA3 ectodomain stimulates nasal RGC axon growth in a concentration dependent way. Moreover, we showed that nasal axons choose growing on EphA3-expressing cells and that EphA3 diminishes the density of interstitial filopodia in nasal RGC axons. Accordingly, in vivo EphA3 ectodomain misexpression directs nasal optic fibers toward the caudal tectum preventing their branching in the rostral tectum. CONCLUSIONS: We demonstrated in vitro and in vivo that EphA3 ectodomain (which is expressed in a decreasing rostro-caudal gradient in the tectum) is necessary for topographic mapping by stimulating the nasal axon growth toward the caudal tectum and inhibiting their branching in the rostral tectum. Furthermore, the ability of EphA3 of stimulating axon growth allows understanding how optic fibers invade the tectum growing throughout this molecular gradient. Therefore, opposing tectal gradients of repellent ephrin-As and of axon growth stimulating EphA3 complement each other to map optic fibers along the rostro-caudal tectal axis

The Emergence and Early Evolution of Biological Carbon-Fixation

The fixation of into living matter sustains all life on Earth, and embeds the biosphere within geochemistry. The six known chemical pathways used by extant organisms for this function are recognized to have overlaps, but their evolution is incompletely understood. Here we reconstruct the complete early evolutionary history of biological carbon-fixation, relating all modern pathways to a single ancestral form. We find that innovations in carbon-fixation were the foundation for most major early divergences in the tree of life. These findings are based on a novel method that fully integrates metabolic and phylogenetic constraints. Comparing gene-profiles across the metabolic cores of deep-branching organisms and requiring that they are capable of synthesizing all their biomass components leads to the surprising conclusion that the most common form for deep-branching autotrophic carbon-fixation combines two disconnected sub-networks, each supplying carbon to distinct biomass components. One of these is a linear folate-based pathway of reduction previously only recognized as a fixation route in the complete Wood-Ljungdahl pathway, but which more generally may exclude the final step of synthesizing acetyl-CoA. Using metabolic constraints we then reconstruct a “phylometabolic” tree with a high degree of parsimony that traces the evolution of complete carbon-fixation pathways, and has a clear structure down to the root. This tree requires few instances of lateral gene transfer or convergence, and instead suggests a simple evolutionary dynamic in which all divergences have primary environmental causes. Energy optimization and oxygen toxicity are the two strongest forces of selection. The root of this tree combines the reductive citric acid cycle and the Wood-Ljungdahl pathway into a single connected network. This linked network lacks the selective optimization of modern fixation pathways but its redundancy leads to a more robust topology, making it more plausible than any modern pathway as a primitive universal ancestral form

Untersuchungen zur Biosynthese von Glycin als Vorstufe von Riboflavin in Ashbya gossypii

The filamentous fungus $\textit{Ashbya gossypii}$ is a vitamin B$_{2}$ overproducer. Enhancement of riboflavin production is known to be achieved by supplementation of the medium with glycine, a precursor of vitamin B$_{2}$ (riboflavin). The present work describes the characterization and deregulation of glycine biosynthetic pathways in $\textit{A. gossypii}$ leading to an improved riboflavin production. In the $\textit{A. gossypii}$ wild type strain ATCC 10895 supplementation with 80 mM glycine lead to an increase in riboflavin production by at least 100 % whereas the growth remained unchanged. Nevertheless only 5 % of the added glycine were consumed during the course of cultivation, which suggested a poor uptake of the amino acid. On unsupplemented media glycine concentration even increased from 1 .9 mM to 3 mM during cultivation. Consequently the effect of glycine on riboflavin production can be attributed to a small net uptake as well as to an inhibition of glycine efflux due to the high extracellular glycine concentration. The glycine biosynthetic enzymes serine hydroxymethyltransferase, threonine aldolase and glutamate glyoxylate aminotransferase were detected in crude extracts of $\textit{A. gossypii}$ with maximum specific activities of 6, 5 and 26 mU/mg protein, respectively. Sucrose density gradient centrifugation of $\textit{A. gossypii}$ organelles showed, that glutamate glyoxylate arninotransferase occurs in the mitochondria of the fungus, thus it is not colocated with isocitrate lyase - the main supplier of glyoxylate - in the peroxisomes. Glycine formation starting from serine and threonine could also be demonstrated $\textit{in vivo}$ using $^{13}$C labelling experiments. Likewise the formation of serine from threonine, which probably proceeds via glycine and therefore means an unwanted loss of glycine, was shown $\textit{in vivo}$. When 70 mnM aminomethylphosphonic acid (AMPS) were added to the culture medium riboflavin production of $\textit{A. gossypii}$ was completely inhibited. Screening on AMPS resistance of riboflavin production lead to the isolation of the strain A.g. AMPS-NM-01. It showed a riboflavin production of 40 mg/g mycelial dry weight (mdw) even in the absence of glycine, which was significantly higher than in the wild type strain (5 mg/g mdw) under the same conditions but resembled wild type riboflavin production in the presence of 80 mM of glycine (30 mg/g mdw). Increased riboflavin production without glycine supplementation suggested a better intracellular availability of glycine in the mutant strain. Nevertheless, even in this case riboflavin production could be increased by glycine supplementation to 95 mg/g mdw. In comparison to the wild type strain serine hydroxymethyltransferase specific activity was significantly reduced from 3 to 1.5 mUlmg protein in the strain A.g. AMPS-NM-01. Therefore the increased riboflavin production of this strain can be explained by a better intracellular availability of glycine conditioned by a reduced loss of glycine for the formation of serine. Using heterologous complementation of a $\textit{Saccharomyces cerevisiae}$ mutant auxotrophic for glycine a GLY1 homologous gene with unknown function was isolated from $\textit{A. gossypii}$. Characterization of the corresponding enzymatic activity showed that the isolated gene as well as the GLY1 gene from $\textit{S. cerevisiae}$ encode a threonine aldolase. In contrast to $\textit{S. cerevisiae}$ the GLY1 knock-out mutant of $\textit{A. gossypii}$ was not auxotrophic for glycine, which demonstrated that threonine aldolase plays only a minor role during glycine biosynthesis of $\textit{A. gossypii}$. GLY1 was overexpressed in $\textit{A. gossypii}$ under the control of the TEF-promotor and -terminator using the expression vector pAG203. In crude extracts of A.g. pAG203GLY1 50 mU/mg protein of threonine aldolase specific activity were detected indicating a tenfold overexpression in comparison to the wild type. When 50 mM threonine was fed to A.g. pAG203GLY1 an increase in riboflavin production from 2 to 16 mg/g mdw was determined, an increase never reached with glycine because of its worse uptake. Threonine was found to be taken up efficiently by this strain. Its conversion to glycine was confirmed by a striking efflux of glycine into the medium. Extracellular glycine correspondingly increased from 2 to 44 mM. The requirement to feed threonine in addition to threonine aldolase overexpression demonstrated a limitation in threonine biosynthesis, which was confirmed by feeding experiments with threonine precursors

Untersuchungen zur Biosynthese von Glycin als Vorstufe von Riboflavin in Ashbya gossypii

Summary in EnglishSIGLEAvailable from TIB Hannover: RA 831(3519) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekDEGerman

Two Eph receptor tyrosine kinase ligands control axon growth and may be involved in the creation of the retinotectal map in the zebrafish

The isolation and characterisation of two zebrafish Eph receptor ligand cDNAs which we have called zfEphL3 and zfEphL4 is described. These genes are expressed in the presumptive midbrain of developing embryos from 6 somites. By 24 hours L3 is expressed throughout the midbrain including the region of the presumptive tectum whereas L4 is strongly expressed in the midbrain caudal to the presumptive tectum. At later stages of development L3 is expressed in a graded fashion throughout the tectum and L4 is maintained at its posterior margin. Growth cone collapse and pathway selection assays demonstrate that both these proteins have a collapse activity for retinal ganglion cells. When faced with a choice of substrate on which to grow, temporal axons from chick retinal ganglion cells selectively avoided membranes from Cos cells transfected with L3, whereas nasal axons did not. Both temporal and nasal axons avoided membranes from Cos cells transfected with L4. The expression patterns together with the functional data suggest that although both ligands may be able to guide retinal ganglion cells axons in vitro, they have different roles in the guidance of retinotectal projections in vivo. The expression of L3 is consistent with a role in the guidance of retinal ganglion cells to their targets on the tectum whereas that of L4 suggests a role in delineating the posterior boundary of the optic tectum

Glycine metabolism in Candida albicans : characterization of the serine hydroxymethyltransferase (SHM1, SHM2) and threonine aldolase (GLY1) genes

Genes encoding the mitochondrial (SHM1) and cytosolic (SHM2) serine hydroxymethyltransferases, and the L-threonine aldolase gene (GLY1) from Candida albicans were cloned and sequenced. All three genes are involved in glycine metabolism. The C. albicans Shm1 protein is 82% identical to that from Saccharomyces cerevisiae and 56% identical to that from Homo sapiens. The corresponding identities for the Shm2 proteins are 68% and 53%. The Gly1 protein shares significant identity with the S. cerevisiae L-threonine aldolase (55%) and also with threonine aldolases from Aeromonas jandiae (36%) and Escherichia coli (36%). Genetic ablation experiments show that GLY1 is a non-essential gene in C. albicans and that L-threonine aldolase plays a lesser role in glycine metabolism than it does in S. cerevisiae. GenBank Accession Nos of the C, albicans SHM1 and SHM2 are AF009965 and AF009966, respectively. Accession No. for C, albicans GLY1 is AF009967. Copyright (C) 2000 John Wiley & Sons, Ltd

Management of Multiple Nitrogen Sources during Wine Fermentation by Saccharomyces cerevisiae

During fermentative growth in natural and industrial environments, Saccharomyces cerevisiae must redistribute the available nitrogen from multiple exogenous sources to amino acids in order to suitably fulfill anabolic requirements. To exhaustively explore the management of this complex resource, we developed an advanced strategy based on the reconciliation of data from a set of stable isotope tracer experiments with labeled nitrogen sources. Thus, quantifying the partitioning of the N compounds through the metabolism network during fermentation, we demonstrated that, contrary to the generally accepted view, only a limited fraction of most of the consumed amino acids is directly incorporated into proteins. Moreover, substantial catabolism of these molecules allows for efficient redistribution of nitrogen, supporting the operative de novo synthesis of proteinogenic amino acids. In contrast, catabolism of consumed amino acids plays a minor role in the formation of volatile compounds. Another important feature is that the alpha - keto acid precursors required for the de novo syntheses originate mainly from the catabolism of sugars, with a limited contribution from the anabolism of consumed amino acids. This work provides a comprehensive view of the intracellular fate of consumed nitrogen sources and the metabolic origin of proteinogenic amino acids, highlighting a strategy of distribution of metabolic fluxes implemented by yeast as a means of adapting to environments with changing and scarce nitrogen resources. IMPORTANCE A current challenge for the wine industry, in view of the extensive competition in the worldwide market, is to meet consumer expectations regarding the sensory profile of the product while ensuring an efficient fermentation process. Understanding the intracellular fate of the nitrogen sources available in grape juice is essential to the achievement of these objectives, since nitrogen utilization affects both the fermentative activity of yeasts and the formation of flavor compounds. However, little is known about how the metabolism operates when nitrogen is provided as a composite mixture, as in grape must. Here we quantitatively describe the distribution through the yeast metabolic network of the N moieties and C backbones of these nitrogen sources. Knowledge about the management of a complex resource, which is devoted to improvement of the use of the scarce N nutrient for growth, will be useful for better control of the fermentation process and the sensory quality of wines

Expression of EphA5 during development of the olfactory nerve pathway in rat

The olfactory neuroepithelium is a highly plastic region of the nervous system that undergoes continual turnover of primary olfactory neurons throughout life. The mechanisms responsible for persistent growth and guidance of primary olfactory axons along the olfactory nerve are unknown. In the present study, we used antibodies against the Eph-related receptor, EphA5, to localise EphA5, and recombinant EDhA5-IgG fusion protein to localise its ligands. We found that although both EphA5 and its ligands were both expressed by primary olfactory neurons within the embryonic olfactory nerve pathway, there was no graded or complementary expression pattern. In contrast, the expression patterns altered postnatally such that primary olfactory neurons expressed the ligands, whereas the second-order olfactory neurons, the mitral cells, expressed EphA5. The role of EphA5 was analysed by blocking EphA5-ligand interactions in explant cultures of olfactory neuroepithelium using anti-EphA5 antibodies and recombinant EphA5. These perturbations reduced neurite outgrowth from explant cultures and suggest that intrafascicular axon repulsion may serve to limit adhesion and optimise conditions for axon growth. (C) 2000 Wiley-Liss, Inc