A dedicated glyceraldehyde-3-phosphate dehydrogenase is involved in the biosynthesis of volatile sesquiterpenes in Trichoderma virens-evidence for the role of a fungal GAPDH in secondary metabolism.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyses the sixth step of glycolysis, and is also known to perform other (moonlighting) activities in animal cells. We have earlier identified an additional GAPDH gene in Trichoderma virens genome. This gene is consistently associated with the vir cluster responsible for biosynthesis of a range of volatile sesquiterpenes in Trichoderma virens. This gene is also associated with an orthologous gene cluster in Aspergillus spp. Both glycolytic GAPDH and the vir cluster-associated GAPDH show more than 80% similarity with essentially conserved NAD+ cofactor- and substrate-binding sites. However, a conserved indel is consistently present only in GAPDH associated with the vir cluster, both in T. virens and Aspergillus spp. Using gene knockout, we demonstrate here that the vir cluster-associated GAPDH is involved in biosynthesis of volatile sesquiterpenes in T. virens. We thus, for the first time, elucidate the non-glycolytic role of a GAPDH in a fungal system, and also prove for the first time that a GAPDH, a primary metabolism protein, is involved in secondary metabolism.

Evolutionary History of the Enzymes Involved in the Calvin-Benson Cycle in Euglenids.

Euglenids are an ancient lineage that may have existed as early as 2 billion years ago. A mere 65 years ago, Melvin Calvin and Andrew A. Benson performed experiments on Euglena gracilis and elucidated the series of reactions by which carbon was fixed and reduced during photosynthesis. However, the evolutionary history of this pathway (Calvin-Benson cycle) in euglenids was more complex than Calvin and Benson could have imagined. The chloroplast present today in euglenophytes arose from a secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga. A long period of evolutionary time existed before this secondary endosymbiotic event took place, which allowed for other endosymbiotic events or gene transfers to occur prior to the establishment of the green chloroplast. This research revealed the evolutionary history of the major enzymes of the Calvin-Benson cycle throughout the euglenid lineage and showed that the majority of genes for Calvin-Benson cycle enzymes shared an ancestry with red algae and/or chromophytes suggesting they may have been transferred to the nucleus prior to the acquisition of the green chloroplast.

A proteomic study of rice cultivar TNG67 and its high aroma mutant SA0420.

Fragrance is a very important economic trait for rice cultivars. To identify the aroma genes in rice, we performed a proteomics analysis of aroma-related proteins between Tainung 67 (TNG67) and its high aroma mutant SA0420. Seventeen of the differentially identified proteins were close related with the aroma phenotype of SA0420. Among them, 9 were found in leaves and 8 were found in grains. One protein (L3) was identified as the chloroplastic glyceraldehyde-3-phosphate dehydrogenase B (OsGAPDHB) which was less abundant in SA0420 than TNG67. Sequence analysis demonstrated that this protein in SA0420 carries a P425S mutation in the C-terminal extension domain, which might hinder the formation of holoenzyme, thereby changing the profile of aroma compounds. The protein profile of OsGAPDHB showed only a weak correlation to its transcription profile. This result indicated that the reduction of OsGAPDHB in SA0420 is regulated by post-translational processes and can only be analyzed by proteomics approach. Transgenic lines suppressing OsGAPDHB through RNAi harbored more fragrance than TNG67 but less than SA0420. With betaine-aldehyde dehydrogenase as the only fragrance gene identified in rice to date, OsGAPDHB may serve as the second protein known to contribute to the aroma phenotype.

Sequence-based molecular phylogenetics and phylogeography of the American box turtles (Terrapene spp.) with support from DNA barcoding.

The classification of the American box turtles (Terrapene spp.) has remained enigmatic to systematists. Previous comprehensive phylogenetic studies focused primarily on morphology. The goal of this study was to re-assess the classification of Terrapene spp. by obtaining DNA sequence data from a broad geographic range and from all four recognized species and 11 subspecies within the genus. Tissue samples were obtained for all taxa except for Terrapene nelsoni klauberi. DNA was extracted, and the mitochondrial DNA (mtDNA) cytochrome b (Cytb) and nuclear DNA (nucDNA) glyceraldehyde-3-phosphate-dehydrogenase (GAPD) genes were amplified via polymerase chain reaction and sequenced. In addition, the mtDNA gene commonly used for DNA barcoding (cytochrome oxidase c subunit I; COI) was amplified and sequenced to calculate pairwise percent DNA sequence divergence comparisons for each Terrapene taxon. The sequence data were analyzed using maximum likelihood and Bayesian phylogenetic inference, a molecular clock, AMOVAs, SAMOVAs, haplotype networks, and pairwise percent sequence divergence comparisons. Terrapene carolina mexicana and T. c. yucatana formed a monophyletic clade with T. c. triunguis, and this clade was paraphyletic to the rest of T. carolina. Terrapene ornata ornata and T. o. luteola lacked distinction phylogenetically, and Terrapene nelsoni was confirmed to be the sister taxon of T. ornata. Terrapene c. major, T. c. bauri, and Terrapene coahuila were not well resolved for some of the analyses. The DNA barcoding results indicated that all taxa were different species (>2% sequence divergence) except for T. c. triunguis - T. c. mexicana and T. o. ornata - T. o. luteola. The results suggest that T. c. triunguis should be elevated to species status (Terrapene mexicana), and mexicana and yucatana should be included in this group as subspecies. In addition, T. o. ornata and T. o. luteola should not be considered separate subspecies. The DNA barcoding data support these recommended taxonomic revisions. Because conservation efforts are typically species-based, these results will be important for facilitating successful conservation management strategies.

HOMSTRAD: recent developments of the Homologous Protein Structure Alignment Database.

HOMSTRAD (http://www-cryst.bioc.cam.ac.uk/ homstrad/) is a collection of protein families, clustered on the basis of sequence and structural similarity. The database is unique in that the protein family sequence alignments have been specially annotated using the program, JOY, to highlight a wide range of structural features. Such data are useful for identifying key structurally conserved residues within the families. Superpositions of the structures within each family are also available and a sensitive structure-aided search engine, FUGUE, can be used to search the database for matches to a query protein sequence. Historically, HOMSTRAD families were generated using several key pieces of software, including COMPARER and MNYFIT, and held in a number of flat files and indexes. A new relational database version of HOMSTRAD, HOMSTRAD BETA (http://www-cryst.bioc.cam. ac.uk/homstradbeta/) is being developed using MySQL. This relational data structure provides more flexibility for future developments, reduces update times and makes data more easily accessible. Consequently it has been possible to add a number of new web features including a custom alignment facility. Altogether, this makes HOMSTRAD and its new BETA version, an excellent resource both for comparative modelling and for identifying distant sequence/structure similarities between proteins.

Subcellular alteration of glyceraldehyde-3-phosphate dehydrogenase in Alzheimer's disease fibroblasts.

The regulation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been implicated both in age-related neurodegenerative disease and in apoptosis. Previous in vitro studies suggest an interaction between GAPDH and the beta-amyloid precursor protein (beta-APP), a protein directly involved in Alzheimer's disease (AD). New studies indicate that GAPDH is a multidimensional protein with diverse membrane, cytoplasmic, and nuclear functions; each is distinct from its role in glycolysis. The nuclear functions of GAPDH include a role in apoptosis that requires its translocation to the nucleus. Accordingly, beta-APP-GAPDH interactions, altering GAPDH structure in vivo, may affect energy generation, inducing hypometabolism, a characteristic AD phenotype. Because GAPDH is a multifunctional protein, pleiotropic effects may also occur in a variety of fundamental cellular pathways in AD cells. This may include unique GAPDH-RNA interactions. We report here the identification of a high-molecular-weight (HMW) GAPDH species present exclusively in the postnuclear fraction of AD cells. The latter is characterized by reduced GAPDH activity. The HMW GAPDH species was not detected in postnuclear age-matched control (AMC) fractions nor in AD whole-cell preparations. Each is characterized by normal GAPDH activity. By definition, the preparation of whole-cell extracts entails the destruction of subcellular structure. The latter findings indicate that the dissociation of the GAPDH protein from the HMW species restores its enzymatic activity. Thus, these results reveal a new, unique intracellular phenotype in AD cells. The functional consequences of subcellular alteration in GAPDH structure in AD cells are considered.

GAPDH gene diversity in spirochetes: a paradigm for genetic promiscuity.

In this study we have determined gap sequences from nine different spirochetes. Phylogenetic analyses of these sequences in the context of all other available eubacterial and a selection of eukaryotic Gap sequences demonstrated that the eubacterial glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene diversity encompasses at least five highly distinct gene families. Within these gene families, spirochetes show an extreme degree of sequence divergence that is probably the result of several lateral gene transfer events between spirochetes and other eubacterial phyla, and early gene duplications in the eubacterial ancestor. A Gap1 sequence from the syphilis spirochete Treponema pallidum has recently been shown to be closely related to GapC sequences from Euglenozoa. Here we demonstrate that several other spirochetal species are part of this cluster, supporting the conclusion that an interkingdom gene transfer from spirochetes to Euglenozoa must have occurred. Furthermore, we provide evidence that the GAPDH genes present in the protists Parabasalia may also be of spirochetal descent.

Molecular characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Phaffia rhodozyma.

The glyceraldehyde-3-phosphate dehydrogenase (GPD; EC1.2.1.12)-encoding gene (gpd) was isolated from a genomic library of Phaffia rhodozyma CBS 6938. Unlike some other eukaryotic organisms the gpd gene is represented by a single copy in P. rhodozyma. The complete nucleotide sequence of the coding, as well as the flanking non-coding regions was determined. The nucleotide sequence of gpd predicted six introns and a polypeptide chain of 339 amino acids. The codon usage in the gpd gene of P. rhodozyma was highly biased and was significantly different from the codon usage in other yeasts. Phylogenetic analysis of different yeasts and filamentous asco- and basidiomycetes gpd sequences indicated that the gpd gene of P. rhodozyma forms a cluster with the corresponding genes of filamentous basidiomycetes.

Evidence for the Heterolobosea from phylogenetic analysis of genes encoding glyceraldehyde-3-phosphate dehydrogenase.

The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme. However, a decade ago, on the basis of detailed ultrastructural resemblances it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea; a dictyostelid, Dictyostelium discoideum; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.

Primary structure and phylogenetic relationships of glyceraldehyde-3-phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates.

Complete nucleotide sequences have been established for two genes (gap1 and gap2) coding for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) homologs in the diplomonad Giardia lamblia. In addition, almost complete sequences of the GAPDH open reading frames were obtained from PCR products for two free-living diplomonad species, Trepomonas agillis and Hexamita inflata, and a parasite of Atlantic salmon, an as yet unnamed species with morphological affinities to Spironucleus. Giardia lamblia gap1 and the genes from the three other diplomonad species show high similarity to each other and to other glycolytic GAPDH genes. All amino-acyl residues known to be highly conserved in this enzyme are also conserved in these sequences. Giardia lamblia gap2 gene is more divergent and its putative translation reveals the presence of a cysteine and serine-rich insertion resembling a metal binding finger. This motif has not yet been noted in other GAPDH molecules. All sequences contain an S-loop signature with characteristics close to those of eukaryotes. In phylogenetic reconstructions based on the derived amino acid sequences with neighbor-joining, parsimony and maximum-likelihood methods the four typical GAPDH sequences of diplomonads cluster into a single clade. Within this clade, G. lambia gap1 shares a common ancestor with the rest of the genes. The latter are more closely related to each other, indicating an early separation of the lineage leading to the genus Giardia from the lineage encompassing the morphologically less differentiated genera, Trepomonas, Hexamita and that of the unnamed species. This result is discordant with the orthogonal evolution of diplomonads suggested on the basis of comparative morphology. In neighbor-joining reconstructions G. lamblia gap2 occupies a variable position, due to its great divergence. In parsimony and maximum likelihood analysis however, it shares a most recent common ancestor with the typical G. lamblia gap1 gene, suggesting that it diverged after the separation of the Giardia lineage. The position of the diplomonad clade in broader phylogenetic reconstructions is firmly within the typical cytosolic glycolytic representatives of GAPDH of eukaryotes.

The evolutionary origin of red algae as deduced from the nuclear genes encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases from Chondrus crispus.

Algae are a heterogeneous group of photosynthetic eukaryotes traditionally separated into three major subdivisions: rhodophytes, chlorophytes, and chromophytes. The evolutionary origin of rhodophytes or red algae and their links to other photosynthetic and nonphotosynthetic eukaryotes have been a matter of much controversy and speculation. Here we present the first cDNAs of nuclear protein genes from red algae: Those encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from Chondrus crispus. A phylogenetic analysis including GAPDH gene sequences from a number of eukaryotic taxa, cyanobacteria, and purple bacteria suggests that chloroplasts and rhodoplasts together form a monophyletic group of cyanobacterial descent and that rhodophytes separated from chlorophytes at about the same time as animals and fungi. The composite GAPDH tree further demonstrates that chloroplast and cytosolic GAPDH genes are closely related to their homologs in cyanobacteria and purple bacteria, respectively, the presumptive ancestors of chloroplasts and mitochondria, thereby firmly establishing the endosymbiotic origin of these nuclear genes and their fixation in eukaryotic cells before the rhodophyte/chlorophyte separation. The present data are in conflict with phylogenetic inferences based on plastid-encoded rbcL sequences supporting a polyphyletic origin of rhodoplasts and chloroplasts. Comparison of rbcL to GAPDH phylogenies suggests that rbcL trees may be misleading because they are composed of branches representing ancient duplicated (paralogous) genes.