Expression pattern of a nuclear encoded mitochondrial arginine-ornithine translocator gene from Arabidopsis

BACKGROUND: Arginine and citrulline serve as nitrogen storage forms, but are also involved in biosynthetic and catabolic pathways. Metabolism of arginine, citrulline and ornithine is distributed between mitochondria and cytosol. For the shuttle of intermediates between cytosol and mitochondria transporters present on the inner mitochondrial membrane are required. Yeast contains a mitochondrial translocator for ornithine and arginine, Ort1p/Arg11p. Ort1p/Arg11p is a member of the mitochondrial carrier family (MCF) essential for ornithine export from mitochondria. The yeast arg11 mutant, which is deficient in Ort1p/Arg11p grows poorly on media lacking arginine. RESULTS: High-level expression of a nuclear encoded Arabidopsis thaliana homolog (AtmBAC2) of Ort1p/Arg11p was able to suppress the growth deficiency of arg11. RT-PCR analysis demonstrated expression of AtmBAC2 in all tissues with highest levels in flowers. Promoter-GUS fusions showed preferential expression in flowers, i.e. pollen, in the vasculature of siliques and in aborted seeds. Variable expression was observed in leaf vasculature. Induction of the promoter was not observed during the first two weeks in seedlings grown on media containing NH(4)NO(3), arginine or ornithine as sole nitrogen sources. CONCLUSION: AtmBAC2 was isolated as a mitochondrial transporter for arginine in Arabidopsis. The absence of expression in developing seeds and in cotyledons of seedlings indicates that other transporters are responsible for storage and mobilization of arginine in seeds

Identification of an Arabidopsis mitochondrial succinate–fumarate translocator

AbstractComplementation of a yeast acr1 mutant carrying a deletion of the succinate/fumarate carrier gene enabled functional identification of a mitochondrial succinate translocator from Arabidopsis thaliana (AtmSFC1). Thus complementation of yeast mutants is applicable also for identification and characterization of organellar transporters. Reverse transcription polymerase chain reaction and promoter-GUS fusion showed expression of AtmSFC1 in 2 day old dark grown seedlings, which declined in cotyledons during further development, consistent with a role in export of fumarate for gluconeogenesis during lipid mobilization at early germination of Arabidopsis seeds. In mature plants, expression was found in developing and germinating pollen, suggesting a role in ethanolic fermentation

Überexpression und Proteinveränderung der Lipasen A and B von Geotrichum candidum CMICC335426

Im wachsenden Interesse an Produkten mit geringerem Gehalt an gesättigten Fettsäuren liegt die Bedeutung der Lipase B von Geotrichum candidum, die eine hohe Spezifität für Fettsäureester von cis-D9 ungesättigten Verbindungen zeigt. Die Lipase B und die hochhomologe Lipase A aus Geotrichum candidum wurden erfolgreich in Saccharomyces cerevisiae exprimiert, mit einer Ausbeute von 6-8 U/(ml Medium). Die Lipasen wurden funktionell und in hoher Ausbeute auch in Pichia pastoris exprimiert. Da ausschließlich die rekombinanten Proteine von dieser Hefe sekretiert werden, liegen die Proteine bereits in nahezu reiner Form im Medium vor. Die Fermentationen der rekombinanten Lipase B wurden unter verschiedenen Kultivierungsbedingungen durchgeführt. Das beste Ergebnis (600.000 U/l Kultur der reinen Lipase B) wurde in einer Hochzelldichtefermentation in billigerem synthetischem Medium erzielt. Die rekombinanten Lipasen A und B wurden hinsichtlich Substratspezifität, Temperatur- und pH-Einfluß charakterisiert. Diese Eigenschaften sind ähnlich wie die der nativen Lipasen A und B von Geotrichum candidum. Ein weiteres Ziel dieser Arbeit war die Erhöhung der Thermostabilität der Lipase B. Dazu wurden Zufallsmutagenese, Punktgerichtete-Mutagenese und Immobilisierung durchgeführt. Durch Zufallsmutagenese und Punktgerichtete-Mutagenese konnten keine Mutanten mit erhöhter Thermostabilität identifiziert werden, während die Immobilisierung auf jeder der verwendeten Matrixen zur Erhöhung der Thermostabilität der Lipase B führte. Die Halbwertszeit des Enzyms bei einer Temperatur von 50 °C konnte von 30 Minuten für die gelöste Lipase auf bis zu mehreren Stunden für die immobilisierte gesteigert werden. Die Überexpression der Lipase B in Pichia pastoris ermöglicht zusammen mit der entwickelten Immobilisierungsmethode eine billige Nutzung des Enzyms in industriellem Maßstab, beispielsweise zur Modifikation der Zusammensetzung von Ölen.The lipase B from Geotrichum candidum, interesting for its peculiar specificity for substrates with a cis-D9 insaturation, and its isoenzyme lipase A were successfully expressed in Saccharomyces cerevisiae. The transformants expressed and secreted the lipases at low levels (6-8 U/ml after 2 days of culture). The lipase genes were also cloned in a vector suitable for expression in the yeast Pichia pastoris. In this expression system, the proteins were functionally expressed and secreted in the culture medium at high levels (50 U/ml after 2 days of culture). The protein of interest was almost the only protein secreted into the medium by Pichia pastoris, thus it was already obtained pure. Fermentations of the Pichia pastoris clone producing lipase B were carried out using different media and in a synthetic medium nearly 600.000 U of pure lipase per liter were reached. The recombinant lipase B was characterised in terms of substrate specificity, pH and temperature stability, activity at different pH values and temperatures. The investigated enzyme properties were comparable to those reported for the native enzyme. To increase the thermostability of lipase B from Geotrichum candidum three different approaches were used: random mutagenesis, site-directed mutagenesis and protein immobilization. With the random- and site-directed mutagenesis approach no mutants with increased thermostability could be identified. Through immobilization on a polypropylene support the half life of the enzyme at 50 °C could be increased from 30 minutes for the soluble lipase to some hours for the immobilized. This work, due to the high expression levels in Pichia pastoris and increased thermostability by immobilization, raises the possibility to develop an industrial application of the specific lipase B from Geotrichum candidum in the modification of the fatty acids profile of oils

A Novel Superfamily of Transporters for Allantoin and Other Oxo Derivatives of Nitrogen Heterocyclic Compounds in Arabidopsis

A wide spectrum of soil heterocyclic nitrogen compounds are potential nutrients for plants. Here, it is shown that Arabidopsis plants are able to use allantoin as sole nitrogen source. By functional complementation of a yeast mutant defective in allantoin uptake, an Arabidopsis transporter, AtUPS1 ( Arabidopsis thaliana ureide permease 1 ), was identified. AtUPS1 belongs to a novel superfamily of plant membrane proteins with five open reading frames in Arabidopsis (identity, 64 to 82%). UPS proteins have 10 putative transmembrane domains with a large cytosolic central domain containing a “Walker A” motif. Transport of 14 C-labeled allantoin by AtUPS1 in yeast exhibited saturation kinetics ( K m ∼ 52 μM), was dependent on Glc and a proton gradient, and was stimulated by acidic pH. AtUPS1 transports uric acid and xanthine, besides allantoin, but not adenine. Protons are cosubstrates in allantoin transport by AtUPS1, as demonstrated by expression in Xenopus laevis oocytes. In plants, AtUPS1 gene expression was dependent on the nitrogen source. Therefore, AtUPS1 presumably is involved in the uptake of allantoin and other purine degradation products when primary sources are limiting

ARAMEMNON, a Novel Database for Arabidopsis Integral Membrane Proteins

A specialized database (DB) for Arabidopsis membrane proteins, ARAMEMNON, was designed that facilitates the interpretation of gene and protein sequence data by integrating features that are presently only available from individual sources. Using several publicly available prediction programs, putative integral membrane proteins were identified among the approximately 25,500 proteins in the Arabidopsis genome DBs. By averaging the predictions from seven programs, approximately 6,500 proteins were classified as transmembrane (TM) candidate proteins. Some 1,800 of these contain at least four TM spans and are possibly linked to transport functions. The ARAMEMNON DB enables direct comparison of the predictions of seven different TM span computation programs and the predictions of subcellular localization by eight signal peptide recognition programs. A special function displays the proteins related to the query and dynamically generates a protein family structure. As a first set of proteins from other organisms, all of the approximately 700 putative membrane proteins were extracted from the genome of the cyanobacterium Synechocystis sp. and incorporated in the ARAMEMNON DB. The ARAMEMNON DB is accessible at the URL http://aramemnon.botanik.uni-koeln.de

N. 6 Schede di catalogo: n. 7.6 Vescovo Lazzaro, Breviario Armeno; n. 7.7 Copista e miniatore ignoti, Evangeliario armeno; n. 7.8 Officina mesopotamica, Mattoni di fondazione con iscrizione cuneiforme; n. 7.18 Ambito veneziano (?), Lavanda dei piedi; n. 7.19 Ambito Italia centrale (?), San Carlo Borromeo e San Filippo Neri; n. 8.6 Attilio Spaccarelli, Coppa con scene dionisiache

La mostra presenta al pubblico – per la prima volta in modo organico – la raccolta vasta e sorprendente che i coniugi statunitensi George Washington Wurts ed Henriette Tower misero insieme a cavallo fra XIX e XX secolo e donarono poi allo Stato italiano, per l’esattezza al museo di Palazzo Venezia, dove tuttora è conservata. Alla base della mostra vi è comunque anche l’idea di restituire il contesto della raccolta Wurts, ovvero quella particolare forma di collezionismo che tra Ottocento e Novecento si legò così intimamente all’Italia, fino a concretizzarsi spesso nella donazione allo Stato di singole opere o di intere raccolte. La mostra illustra le dinamiche del collezionismo, soprattutto anglo-americano, e del mercato internazionale, sullo sfondo dei radicali cambiamenti vissuti in quegli anni dalla giovane nazione italiana e dalla sua nuova capitale, Roma. La costruzione del Vittoriano, iniziato nel 1885 e inaugurato nel 1911 nell’occasione dell’Esposizione che celebrava il cinquantenario dell’Unità d’Italia, diviene l’emblema che caratterizza la città all’alba del Novecento