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Arabidopsis cell walls contain large amounts of pectins and hemicelluloses, which are predominantly synthesized via the common precursor UDP-glucuronic acid. The major enzyme for the formation of this nucleotide-sugar is UDP-glucose dehydrogenase, catalysing the irreversible oxidation of UDP-glucose into UDP-glucuronic acid. Four functional gene family members and one pseudogene are present in the Arabidopsis genome, and they show distinct tissue-specific expression patterns during plant development. The analyses of reporter gene lines indicate gene expression of UDP-glucose dehydrogenases in growing tissues. The biochemical characterization of the different isoforms shows equal affinities for the cofactor NAD+ (~40 µM) but variable affinities for the substrate UDP-glucose (120–335 µM) and different catalytic constants, suggesting a regulatory role for the different isoforms in carbon partitioning between cell wall formation and sucrose synthesis as the second major UDP-glucose-consuming pathway. UDP-glucose dehydrogenase is feedback inhibited by UDP-xylose. The relatively (compared with a soybean UDP-glucose dehydrogenase) low affinity of the enzymes for the substrate UDP-glucose is paralleled by the weak inhibition of the enzymes by UDP-xylose. The four Arabidopsis UDP-glucose dehydrogenase isoforms oxidize only UDP-glucose as a substrate. Nucleotide-sugars, which are converted by similar enzymes in bacteria, are not accepted as substrates for the Arabidopsis enzymes.
Schnütgen, Frank ; Hansen, Jens ; De-Zolt, Silke ; Horn, Carsten ; Lutz, Marcus ; Floss, Thomas ; Wurst, Wolfgang ; Ruiz Noppinger, Patricia ; Melchner, Harald von
Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.
Comparative studies suggest that at least some bird species have evolved mental skills similar to those found in humans and apes. This is indicated by feats such as tool use, episodic-like memory, and the ability to use one´s own experience in predicting the behavior of conspecifics. It is, however, not yet clear whether these skills are accompanied by an understanding of the self. In apes, self-directed behavior in response to a mirror has been taken as evidence of self-recognition. We investigated mirror-induced behavior in the magpie, a songbird species from the crow family. As in apes, some individuals behaved in front of the mirror as if they were testing behavioral contingencies. When provided with a mark, magpies showed spontaneous mark-directed behavior. Our findings provide the first evidence of mirror self-recognition in a non-mammalian species. They suggest that essential components of human self-recognition have evolved independently in different vertebrate classes with a separate evolutionary history.
Taylor, Alexander B. ; Meyer, Britta ; Leal, Belinda Z. ; Kötter, Peter ; Schirf, Virgil ; Demeler, Borries ; Hart, P. John ; Entian, Karl-Dieter ; Wöhnert, Jens
Ribosome biogenesis in eukaryotes requires the participation of a large number of ribosome assembly factors. The highly conserved eukaryotic nucleolar protein Nep1 has an essential but unknown function in 18S rRNA processing and ribosome biogenesis. In Saccharomyces cerevisiae the malfunction of a temperature-sensitive Nep1 protein (nep1-1ts) was suppressed by the addition of S-adenosylmethionine (SAM). This suggests the participation of Nep1 in a methyltransferase reaction during ribosome biogenesis. In addition, yeast Nep1 binds to a 6-nt RNA-binding motif also found in 18S rRNA and facilitates the incorporation of ribosomal protein Rps19 during the formation of pre-ribosomes. Here, we present the X-ray structure of the Nep1 homolog from the archaebacterium Methanocaldococcus jannaschii in its free form (2.2 Å resolution) and bound to the S-adenosylmethionine analog S-adenosylhomocysteine (SAH, 2.15 Å resolution) and the antibiotic and general methyltransferase inhibitor sinefungin (2.25 Å resolution). The structure reveals a fold which is very similar to the conserved core fold of the SPOUT-class methyltransferases but contains a novel extension of this common core fold. SAH and sinefungin bind to Nep1 at a preformed binding site that is topologically equivalent to the cofactor-binding site in other SPOUT-class methyltransferases. Therefore, our structures together with previous genetic data suggest that Nep1 is a genuine rRNA methyltransferase.
Many new gene copies emerged by gene duplication in hominoids, but little is known with respect to their functional evolution. Glutamate dehydrogenase (GLUD) is an enzyme central to the glutamate and energy metabolism of the cell. In addition to the single, GLUD-encoding gene present in all mammals (GLUD1), humans and apes acquired a second GLUD gene (GLUD2) through retroduplication of GLUD1, which codes for an enzyme with unique, potentially brain-adapted properties. Here we show that whereas the GLUD1 parental protein localizes to mitochondria and the cytoplasm, GLUD2 is specifically targeted to mitochondria. Using evolutionary analysis and resurrected ancestral protein variants, we demonstrate that the enhanced mitochondrial targeting specificity of GLUD2 is due to a single positively selected glutamic acid-to-lysine substitution, which was fixed in the N-terminal mitochondrial targeting sequence (MTS) of GLUD2 soon after the duplication event in the hominoid ancestor ~18–25 million years ago. This MTS substitution arose in parallel with two crucial adaptive amino acid changes in the enzyme and likely contributed to the functional adaptation of GLUD2 to the glutamate metabolism of the hominoid brain and other tissues. We suggest that rapid, selectively driven subcellular adaptation, as exemplified by GLUD2, represents a common route underlying the emergence of new gene functions.
Koppers-Lalic, Danijela ; Verweij, Marieke C. ; Lipinska, Andrea D. ; Wang, Ying ; Quinten, Edwin ; Reits, Eric A. ; Koch, Joachim ; Loch, Sandra ; Marcondes Rezende, Marisa ; Daus, Franz ; Bienkowska-Szewczyk, Krystyna ; Osterrieder, Nikolaus ; Mettenleiter, Thomas C. ; Heemskerk, Mirjam H. M. ; Tampé, Robert ; Neefjes, Jacques J. ; Chowdhury, Shafiqul I. ; Ressing, Maaike E. ; Rijsewijk, Frans A. M. ; Wiertz, Emmanuel J. H. J.
Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms.
Nagata, Takashi ; Suzuki, Sakura ; Endo, Ryuta ; Shirouzu, Mikako ; Terada, Takaho ; Inoue, Makoto ; Kigawa, Takanori ; Kobayashi, Naohiro ; Güntert, Peter ; Tanaka, Akiko ; Hayashizaki, Yoshihide ; Muto, Yutaka ; Yokoyama, Shigeyuki
The degradation of the poly(A) tail is crucial for posttranscriptional gene regulation and for quality control of mRNA. Poly(A)-specific ribonuclease (PARN) is one of the major mammalian 3’ specific exo-ribonucleases involved in the degradation of the mRNA poly(A) tail, and it is also involved in the regulation of translation in early embryonic development. The interaction between PARN and the m7GpppG cap of mRNA plays a key role in stimulating the rate of deadenylation. Here we report the solution structures of the cap-binding domain of mouse PARN with and without the m7GpppG cap analog. The structure of the cap-binding domain adopts the RNA recognition motif (RRM) with a characteristic a-helical extension at its C-terminus, which covers the b-sheet surface (hereafter referred to as PARN RRM). In the complex structure of PARN RRM with the cap analog, the base of the N7-methyl guanosine (m7G) of the cap analog stacks with the solvent-exposed aromatic side chain of the distinctive tryptophan residue 468, located at the C-terminal end of the second b-strand. These unique structural features in PARN RRM reveal a novel cap-binding mode, which is distinct from the nucleotide recognition mode of the canonical RRM domains.
Hiß, Jan Alexander ; Przyborski, Jude Marek ; Schwarte, Florian ; Lingelbach, Klaus ; Schneider, Gisbert
We performed a bioinformatical analysis of protein export elements (PEXEL) in the putative proteome of the malaria parasite Plasmodium falciparum. A protein family-specific conservation of physicochemical residue profiles was found for PEXEL-flanking sequence regions. We demonstrate that the family members can be clustered based on the flanking regions only and display characteristic hydrophobicity patterns. This raises the possibility that the flanking regions may contain additional information for a family-specific role of PEXEL. We further show that signal peptide cleavage results in a positional alignment of PEXEL from both proteins with, and without, a signal peptide.
Andreone, Franco ; Carpenter, Angus I. ; Cox, Neil ; du Preez, Louis ; Freeman, Karen ; Furrer, Samuel ; Garcia, Gerardo ; Glaw, Frank ; Glos, Julian ; Knox, David ; Köhler, Jörn ; Mendelson III, Joseph R. ; Mercurio, Vincenzo ; Mittermeier, Russell A. ; Moore, Robin D. ; Rabibisoa, Nirhy H. C. ; Randriamahazo, Herilala ; Randrianasolo, Harison ; Raminosoa, Noromalala Rasoamampionona ; Ramilijaona, Olga Ravoahangimalala ; Raxworthy, Christopher J. ; Vallan, Denis ; Vences, Miguel ; Vieites, David R. ; Weldon, Ché
Hummel, Hans E. ; Dinnesen, Sven ; Nedelev, Tenio ; Modic, Spela ; Urek, Gregor ; Ulrichs, Christian
Diabrotica virgifera virgifera LeConte, in its original North American habitat also known as western corn rootworm beetle, actively continues its expansion to new territories and uses Homo sapiens as its prime vector. It took only 15 years to spread to and occupy the southeastern and central parts of Europe, so far with the exception of Denmark where it has not been documented as of 2007. Economic thresholds have been reached and surpassed only in Southeast European countries like Slovakia, Hungary, Serbia, Eastern Croatia, Romania and Northern Italy. But both, the area affected and the severity of symptoms are increasing. Model calculations by a number of authors (Baufeld & Enzian, 2005 a and b; Hongmei Li & al. 2006, CLIMEX model) indicate a definitive propensity of D. v. virgifera to expand its currently occupied territory to regions with moderate temperatures and Zea mays cultivation. East Africa and Eastern Asia are included in the list of potential candidates for future inadvertent introduction. In most discussions it is tacitly and erroneously assumed that Z. mays is the only or the only important host of D. v. virgifera. Our recent observations in Eastern Slovenia on the oil pumpkin Cucurbita pepo indicate, however, that this simplifying assumption is notlonger strictly valid. It has to be modified in light of new evidence. Here, we report a few field experiments conducted in August of 2006 clarifying the host status of C. pepo in a European country.