Prediction of functional class of novel plant proteins by a statistical learning method

In plant genomes, the function of a substantial percentage of the putative protein-coding open reading frames (ORFs) is unknown. These ORFs have no significant sequence similarity to known proteins, which complicates the task of functional study of these proteins. Efforts are being made to explore methods that are complementary to, or may be used in combination with, sequence alignment and clustering methods. A web-based protein functional class prediction software, SVMProt, has shown some capability for predicting functional class of distantly related proteins. Here the usefulness of SVMProt for functional study of novel plant proteins is evaluated. To test SVMProt, 49 plant proteins (without a sequence homolog in the Swiss-Prot protein database, not in the SVMProt training set, and with functional indications provided in the literature) were selected from a comprehensive search of MEDLINE abstracts and Swiss-Prot databases in 1999-2004. These represent unique proteins the function of which, at present, cannot be confidently predicted by sequence alignment and clustering methods. The predicted functional class of 31 proteins was consistent, and that of four other proteins was weakly consistent, with published functions. Overall, the functional class of 71.4% of these proteins was consistent, or weakly consistent, with functional indications described in the literature. SVMProt shows a certain level of ability to provide useful hints about the functions of novel plant proteins with no similarity to known proteins.     

Identification of an EcoRI restriction site for a rapid and precise determination of beta-asarone-free Acorus calamus cytotypes

Calamus (Acorus calamus L., Araceae) is an aromatic herb, indigenous to Central Asia and Eastern Europe. The fragrant oils obtained by alcoholic extraction of the rhizome are mainly used in the pharmaceutical and oenological industries. Nevertheless, the occurrence of beta-asarone [(Z)-1,2,4-trimethoxy-5-prop-1-enyl-benzene] limits the possibility of its use due to the carcinogenic properties of this compound. The aim of this work was to identify a diploid beta-asarone-free A. calamus by using chemical and molecular approaches. For these purposes alcoholic extracts of both diploid and triploid A. calamus were analyzed by gas chromatography-mass spectrometry (GC-MS) and comparison of the 700 bp sequence of the non-transcribed spacer (NTS) in the 5S-rRNA gene was also performed. Alcoholic extracts of the triploid A. calamus were characterized by a higher percentage of beta-asarone (11%), which was the main compound, followed by higher percentages of camphene (2.27%), E-beta-ocimene (3.28%), camphor (1.54%), calarene (1.42%), alpha-selinene (5.02%) and tau-cadinol (2.00%), when compared to the diploid A. calamus. The latter had higher percentages of iso-shyobunone (8.62%), beta-sesquiphellandrene (3.28%), preiso calamendiol (22.81%) and acorone (26.33%), and completely lacked of beta-asarone. The 5S-rRNA spacer region of both diploid and triploid A. calamus were amplified by PCR using a pair of primers located at the 3' and 5' ends of the coding sequence of 5S-rRNA gene. The resulting PCR products (about 700 bp) were gel purified, subcloned into pGEM-T Easy vector and sequenced. By aligning the isolated nucleotide sequences of the two varieties and the sequences from different A. calamus chemotypes present in Genbank, sequence diversities were found in the spacer region. Furthermore, the PCR products were digested by using EcoRI. The restriction profile of the spacer domain resulted different for the two cytotypes. Along with chemical analysis of alcoholic extracts, sequence analysis coupled to restriction mapping was demonstrated to represent a powerful tool to distinguish the A. calamus diploid cytotype from the others. The security and effective usage of the diploid beta-asarone-free A. calamus was also discussed.     

Selenium uptake,translocation, assimilation and metabolic fate in plants

The chemical and physical resemblance between selenium (Se) and sulfur (S) establishes that both these elements share common metabolic pathways in plants. The presence of isologous Se and S compounds indicates that these elements compete in biochemical processes that affect uptake, translocation and assimilation throughout plant development. Yet, minor but crucial differences in reactivity and other metabolic interactions infer that some biochemical processes involving Se may be excluded from those relating to S. This review examines the current understanding of physiological and biochemical relationships between S and Se metabolism by highlighting their similarities and differences in relation to uptake, transport and assimilation pathways as observed in Se hyperaccumulator and non-accumulator plant species. The exploitation of genetic resources used in bioengineering strategies of plants is illuminating the function of sulfate transporters and key enzymes of the S assimilatory pathway in relation to Se accumulation and final metabolic fate. These strategies are providing the basic framework by which to resolve questions relating to the essentiality of Se in plants and the mechanisms utilized by Se hyperaccumulators to circumvent toxicity. In addition, such approaches may assist in the future application of genetically engineered Se accumulating plants for environmental renewal and human health objectives.     

Analysis of p300 acetyltransferase substrate specificity by MALDI TOF mass spectrometry

Acetyltransferase enzymes target specific lysine residues in substrate proteins. While the list of histone and nonhistone substrates is growing, the mechanisms of substrate selection remain unclear. Here, we describe a mass spectrometric approach to examine the site selection of the acetyltransferase p300 in the HIV-1 protein Tat. Tat is acetylated by p300 at a single lysine (K50) within its basic RNA-binding domain. To determine the sequence requirements for K50 recognition within this domain, we synthesized mixtures of "degenerated" Tat peptides, in which one of the surrounding residues was substituted by all proteinogenic amino acids. Peptide mixtures were assembled based on nonoverlapping peptide masses and acetylated by p300 in a standard in vitro acetylation reaction. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified amino acid substitutions that prevented acetylation by p300. This approach represents a fast and comprehensive screening method that was applied to the six surrounding residues of K50 in Tat. It can be applied to any known acetyltransferase substrate and might help to define consensus recognition sequences for individual acetyltransferase enzymes.     

Multiple events of horizontal transfer of the Minos transposable element between Drosophila species

In this study the Minos element was analyzed in 26 species of the repleta group and seven species of the saltans group of the genus Drosophila. The PCR and Southern blot analysis showed a wide occurrence of the Minos transposable element among species of the repleta and the saltans groups and also a low number of insertions in both genomes. Three different analyses, nucleotide divergence, historical associations, and comparisons between substitution rates (d(N) and d(S)) of Minos and Adh host gene sequences, suggest the occurrence of horizontal transfer between repleta and saltans species. These data reinforce and extend the Arca and Savakis [Genetica 108 (2000) 263] results and suggest five events of horizontal transfer to explain the present Minos distribution: between D. saltans and the ancestor of the mulleri and the mojavensis clusters; between D. hydei and the ancestor of the mulleri and the mojavensis clusters; between D. mojavensis and D. aldrichi; between D. buzzatii and D. serido; and between D. spenceri and D. emarginata. An alternative explanation would be that repeated events of horizontal transfer involving D. hydei, which is a cosmopolitan species that diverged from the others repleta species as long as 14Mya, could have spread Minos within the repleta group and to D. saltans. The data presented in this article support a model in which distribution of Minos transposon among Drosophila species is determined by horizontal transmission balanced by vertical inactivation and extinction.     

High level expression of a recombinant acid phytase gene in Pichia pastoris

AIMS: To achieve high phytase yield with improved enzymatic activity in Pichia pastoris. METHODS AND RESULTS: The 1347-bp phytase gene of Aspergillus niger SK-57 was synthesized using a successive polymerase chain reaction and was altered by deleting intronic sequences, optimizing codon usage and replacing its original signal sequence with a synthetic signal peptide (designated MF4I) that is a codon-modified Saccharomyces cerevisiae mating factor alpha-prepro-leader sequence. The gene constructs containing wild type or modified phytase gene coding sequences under the control of the highly-inducible alcohol oxidase gene promoter with the MF4I- or wild type alpha-signal sequence were used to transform Pichia pastoris. The P. pastoris strain that expressed the modified phytase gene (phyA-sh) with MF4I sequence produced 6.1 g purified phytase per litre of culture fluid, with the phytase activity of 865 U ml(-1). The expressed phytase varied in size (64, 67, 87, 110 and 120 kDa), but could be deglycosylated to produce a homogeneous 64 kDa protein. The recombinant phytase had two pH optima (pH 2.5 and pH 5.5) and an optimum temperature of 60 degrees C. CONCLUSIONS: The P. pastoris strain with the genetically engineered phytase gene produced 6.1 g l(-1) of phytase or 865 U ml(-1) phytase activity, a 14.5-fold increase compared with the P. pastoris strain with the wild type phytase gene. SIGNIFICANCE AND IMPACT OF THE STUDY: The P. pastoris strain expressing the modified phytase gene with the MF4I signal peptide showed great potential as a commercial phytase production system.