Primary structure of glyceraldehyde-3-phosphate dehydrogenase deduced from the nucleotide sequence of the thermophilic archaebacterium Methanothermus fervidus

The gene for the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from the thermophilic methanogenic archaebacterium Methanothermus fervidus (growth optimum at 84 degrees C) was cloned in Escherichia coli and the nucleotide sequence was determined. A striking preference for adenine and thymidine bases was found in the gene, which is in agreement with the low G + C content of the M. fervidus DNA. The deduced amino acid sequence indicates an Mr of 37,500 for the protein subunit. Alignment with the amino acid sequences of GAPDHs from other organisms shows that the archaebacterial GAPDH is homologous to the respective eubacterial and eukaryotic enzymes, but the similarity between the archaebacterial enzyme and the eubacterial or eukaryotic GAPDHs is much less than that between the latter two.     

Primary structure and eubacterial relationships of the pyruvate:Ferredoxin oxidoreductase of the amitochondriate eukaryoteTrichomonas vaginalis

In the eukaryotic unicellular organismTrichomonas vaginalis a key step of energy metabolism, the oxidative decarboxylation of pyruvate with the formation of acetyl-CoA, is catalyzed by the iron-sulfur protein pyruvate:ferredoxin oxidoreductase (PFO) and not by the almost-ubiquitous pyruvate dehydrogenase multienzyme complex. This enzyme is localized in the hydrogenosome, an organelle bounded by a double membrane. PFO and its closely related homolog, pyruvate: flavodoxin oxidoreductase, are enzymes found in a number of archaebacteria and eubacteria. The presence of these enzymes in eukaryotes is restricted, however, to a few amitochondriate groups. To gain more insight into the evolutionary relationships ofT. vaginalis PFO we determined the primary structure of its two genes (pfoA andpfoB). The deduced amino acid sequences showed 95% positional identity. Motifs implicated in related enzymes in liganding the Fe-S centers and thiamine pyrophosphate were well conserved. TheT. vaginalis PFOs were found to be homologous to eubacterial pyruvate: flavodoxin oxidoreductases and showed about 40% amino acid identity to these enzymes over their entire length. Lack of eubacterial PFO sequences precluded a comparison.pfoA andpfoB revealed a greater distance from related enzymes of Archaebacteria. The conceptual translation of the nucleotide sequences predicted an amino-terminal pentapeptide not present in the mature protein. This processed leader sequence was similar to but shorter than leader sequences noted in other hydrogenosomal proteins. These sequences are assumed to be involved in organellar targeting and import. The results underscore the unusual characteristics ofT. vaginalis metabolism and of their hydrogenosomes. They also suggest that in its energy metabolismT. vaginalis is closer to eubacteria than archaebacteria.Abbreviations PCR DNA polymerase chain reaction - PDH pyruvate dehydrogenase - PFO pyruvate:ferredoxin oxidoreductase - TPP thiamine pyrophosphate Correspondence to: M. Müller     

Phylogenetic Relationships of the Glycolytic Enzyme, Glyceraldehyde-3-Phosphate Dehydrogenase, from Parabasalid Flagellates

Over 90% of the open reading frame of gap genes for glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH; EC 1.2.1.12) was obtained with PCR from five species of Parabasala. With gap1 from Trichomonas vaginalis obtained earlier, the data include two sequences each for three species. All sequences were colinear with T. vaginalis gap1 and shared with it as a synapomorphy a 10- to 11-residue insertion not found in any other gap and an S-loop with characteristic features of eubacterial GAPDH. All residues known to be highly conserved in this enzyme were present. The parabasalid sequences formed a robust monophyletic group in phylogenetic reconstructions with distance-based, maximum-parsimony, and maximum-likelihood methods. The two genes of the amphibian commensal, Trichomitus batrachorum, shared a common ancestor with the rest, which separate into two well-supported lineages. T. vaginalis and Tetratrichomonas gallinarum (both representatives of Trichomonadinae) formed one, while Monocercomonas sp. and Tritrichomonas foetus formed the other. These data agreed with and/or were close to published reconstructions based on other macromolecules. They did not support the ancestral position of Monocercomonas sp. proposed on the basis of morphological characteristics but confirmed an early emergence of Trichomitus batrachorum. The sequence pairs obtained from three species indicated either gene duplications subsequent to the divergence of the corresponding lineages or a strong gene conversion later in these lineages. The parabasalid clade was a robust part of the eubacterial radiation of GAPDH and showed no relationships to the clade that contained all other eukaryotic gap genes. The data clearly reveal that the members of this lineage use in their glycolytic pathway a GAPDH species with properties and an evolutionary history that are unique among all eukaryotes studied so far. Received: 28 April 1997 / Accepted: 14 October 1997     

Comparative Analysis of the Ribosomal Componentsof the Hydrogenosome-Containing Protist, Trichomonas vaginalis

The ribosomes of the amitochondriate but hydrogenosome-containing protist lineage, the trichomonads, have previously been reported to be prokaryotic or primitive eukaryotic, based on evidence that they have a 70S sedimentation coefficient and a small number of proteins, similar to prokaryotic ribosomes. In order to determine whether the components of the trichomonad ribosome indeed differ from those of typical eukaryotic ribosomes, the ribosome of a representative trichomonad, Trichomonas vaginalis, was characterized. The sedimentation coefficient of the T. vaginalis ribosome was smaller than that of Saccharomyces cerevisiae and larger than that of Escherichia coli. Based on two-dimensional PAGE analysis, the number of different ribosomal proteins was estimated to be approximately 80. This number is the same as those obtained for typical eukaryotes (approximately 80) but larger than that of E. coli (approximately 55). N-Terminal amino acid sequencing of 18 protein spots and the complete sequences of 4 ribosomal proteins as deduced from their genes revealed these sequences to display typical eukaryotic features. Phylogenetic analyses of the five ribosomal proteins currently available also clearly confirmed that the T. vaginalis sequences are positioned within a eukaryotic clade. Comparison of deduced secondary structure models of the small and large subunit rRNAs of T. vaginalis with those of other eukaryotes revealed that all helices commonly found in typical eukaryotes are present and conserved in T. vaginalis, while variable regions are shortened or lost. These lines of evidence demonstrate that the T. vaginalis ribosome has no prokaryotic or primitive eukaryotic features but is clearly a typical eukaryotic type.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Heat Shock Protein 70 Family: Multiple Sequence Comparisons, Function, and Evolution

The heat shock protein 70 kDa sequences (HSP70) are of great importance as molecular chaperones in protein folding and transport. They are abundant under conditions of cellular stress. They are highly conserved in all domains of life: Archaea, eubacteria, eukaryotes, and organelles (mitochondria, chloroplasts). A multiple alignment of a large collection of these sequences was obtained employing our symmetric-iterative ITERALIGN program (Brocchieri and Karlin 1998). Assessments of conservation are interpreted in evolutionary terms and with respect to functional implications. Many archaeal sequences (methanogens and halophiles) tend to align best with the Gram-positive sequences. These two groups also miss a signature segment [about 25 amino acids (aa) long] present in all other HSP70 species (Gupta and Golding 1993). We observed a second signature sequence of about 4 aa absent from all eukaryotic homologues, significantly aligned in all prokaryotic sequences. Consensus sequences were developed for eight groups [Archaea, Gram-positive, proteobacterial Gram-negative, singular bacteria, mitochondria, plastids, eukaryotic endoplasmic reticulum (ER) isoforms, eukaryotic cytoplasmic isoforms]. All group consensus comparisons tend to summarize better the alignments than do the individual sequence comparisons. The global individual consensus ``matches' 87% with the consensus of consensuses sequence. A functional analysis of the global consensus identifies a (new) highly significant mixed charge cluster proximal to the carboxyl terminus of the sequence highlighting the hypercharge run EEDKKRRER (one-letter aa code used). The individual Archaea and Gram-positive sequences contain a corresponding significant mixed charge cluster in the location of the charge cluster of the consensus sequence. In contrast, the four Gram-negative proteobacterial sequences of the alignment do not have a charge cluster (even at the 5% significance level). All eukaryotic HSP70 sequences have the analogous charge cluster. Strikingly, several of the eukaryotic isoforms show multiple mixed charged clusters. These clusters were interpreted with supporting data related to HSP70 activity in facilitating chaperone, transport, and secretion function. We observed that the consensus contains only a single tryptophan residue and a single conserved cysteine. This is interpreted with respect to the target rule for disaggregating misfolded proteins. The mitochondrial HSP70 connections to bacterial HSP70 are analyzed, suggesting a polyphyletic split of Trypanosoma and Leishmania protist mitochondrial (Mt) homologues separated from Mt-animal/fungal/plant homologues. Moreover, the HSP70 sequences from the amitochondrial Entamoeba histolytica and Trichomonas vaginalis species were analyzed. The E. histolytica HSP70 is most similar to the higher eukaryotic cytoplasmic sequences, with significantly weaker alignments to ER sequences and much diminished matching to all eubacterial, mitochondrial, and chloroplast sequences. This appears to be at variance with the hypothesis that E. histolytica rather recently lost its mitochondrial organelle. T. vaginalis contains two HSP70 sequences, one Mt-like and the second similar to eukaryotic cytoplasmic sequences suggesting two diverse origins. Received: 29 January 1998 / Accepted: 14 May 1998     

Comparison and Evolutionary Analysis of the Glycosomal Glyceraldehyde-3-Phosphate Dehydrogenase from Different Kinetoplastida

In this work, we present the sequences and a comparison of the glycosomal GAPDHs from a number of Kinetoplastida. The complete gene sequences have been determined for some species (Crithidia fasciculata, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas sp), whereas for other species (Trypanosoma brucei gambiense, Trypanosoma congolense, Trypanosoma vivax, and Leishmania major), only partial sequences have been obtained by PCR amplification. The structure of all available glycosomal GAPDH genes was analyzed in detail. Considerable variations were observed in both their nucleotide composition and their codon usage. The GC content varies between 64.4% in L. seymouri and 49.5% in the previously sequenced GAPDH gene from Trypanoplasma borreli. A highly biased codon usage was found in C. fasciculata, with only 34 triplets used, whereas in T. borreli 57 codons were employed. No obvious correlation could be observed between the codon usage and either the nucleotide composition or the level of gene expression. The glycosomal GAPDH is a very well-conserved enzyme. The maximal overall difference observed in the amino acid sequences is only 25%. Specific insertions and extensions are retained in all sequences. The residues involved in catalysis, substrate, and inorganic phosphate binding are fully conserved, whereas some variability is observed in the cofactor-binding pocket. The implications of these data for the design of new trypanocidal drugs targeted against GAPDH are discussed. All available gene and amino acid sequences of glycosomal GAPDHs were used for a phylogenetic analysis. The division of the Kinetoplastida into two suborders, Bodonina and Trypanosomatina, was well supported. Within the letter group, the Trypanosoma species appeared to be monophyletic, whereas the other trypanosomatids form a second clade. Received: 23 February 1998/Accepted: 26 March 1998     

The Pyrophosphate-Dependent Phosphofructokinase of the Protist, Trichomonas vaginalis, and the Evolutionary Relationships of Protist Phosphofructokinases

The pyrophosphate-dependent phosphofructokinase (PP_i-PFK) of the amitochondriate protist Trichomonas vaginalis has been purified. The enzyme is a homotetramer of about 50 kDa subunits and is not subject to allosteric regulation. The protein was fragmented and a number of peptides were sequenced. Based on this information a PCR product was obtained from T. vaginalis gDNA and used to isolate corresponding cDNA and gDNA clones. Southern analysis indicated the presence of five genes. One open reading frame (ORF) was completely sequenced and for two others the 5′ half of the gene was determined. The sequences were highly similar. The complete ORF corresponded to a polypeptide of about 46 kDa. All the peptide sequences obtained were present in the derived sequences. The complete ORF was highly similar to that of other PFKs, primarily in its amino-terminal half. The T. vaginalis enzyme was most similar to PP_i-PFK of the mitochondriate heterolobosean, Naegleria fowleri. Most of the residues shown or assumed to be involved in substrate binding in other PP_i-PFKs were conserved in the T. vaginalis enzyme. Direct comparison and phylogenetic reconstruction revealed a significant divergence among PP_i-PFKs and related enzymes, which can be assigned to at least four distantly related groups, three of which contain enzymes of protists. The separation of these groups is supported with a high percentage of bootstrap proportions. The short T. vaginalis PFK shares a most recent common ancestor with the enzyme from N. fowleri. This pair is clearly separated from a group comprising the long (>60-kDa) enzymes from Giardia lamblia, Entamoeba histolytica pfk2, the spirochaetes Borrelia burgdorferi and Trepomena pallidum, as well as the α- and β-subunits of plant PP_i-PFKs. The third group (``X') containing protist sequences includes the glycosomal ATP-PFK of Trypanosoma brucei, E. histolytica pfk1, and a second sequence from B. burgdorferi. The fourth group (``Y') comprises cyanobacterial and high-G + C, Gram-positive eubacterial sequences. The well-studied PP_i-PFK of Propionibacterium freudenreichii is highly divergent and cannot be assigned to any of these groups. These four groups are well separated from typical ATP-PFKs, the phylogenetic analysis of which confirmed relationships established earlier. These findings indicate a complex history of a key step of glycolysis in protists with several early gene duplications and possible horizontal gene transfers. Received: 5 December 1997 / Accepted: 18 March 1998     

Trichomonas vaginalis and early evolving DNA and protein sequences of the CDC2/28 protein kinase family

The human sexually transmittted parasite Trichomonas vaginalis is a representative of one of the three earliest evolving eukaryotic lineages. We investigated whether T. vaginalis has DNA sequences and peptides related to cell division control molecules universal among yeasts and higher eukaryotes. A T. vaginalis ceil division control (CDC2/28) homologue was amplified by the polymerase chain reaction and sequenced. The absolute similarity with other CDC2/28 genes was 47%, with conservative replacement similarity of 67%. Western blots demonstrated a single T. vaginalis peptide reactive with antiserum to the PSTAIRE peptide, an expressed component of CDC2/28 genes in higher eukaryotes. Although eukaryotic, T. vaginalis has properties similar to those of bacteria and is the earlist evolving eukaryote reported to possess CDC2/28 DNA and peptide homologues. These observations suggest that the molecular origins of cell division control in eukaroytes preceded mitochondria, 28S ribosomes and regulated glycolysis.     

The sequence and organization of the core histone H3 and H4 genes in the early branching amitochondriate protistTrichomonas vaginalis

Among the unicellular protists, several of which are parasitic, some of the most divergent eukaryotic species are found. The evolutionary distances between protists are so large that even slowly evolving proteins like histones are strongly divergent. In this study we isolated cDNA and genomic histone H3 and H4 clones fromTrichomonas vaginalis. Two histone H3 and three histone H4 genes were detected on three genomic clones with one complete H3 and two complete H4 sequences. H3 and H4 genes were divergently transcribed with very short intergenic regions of only 194 bp, which containedT. vaginalis-specific as well as histone-specific putative promoter elements. Southern blot analysis showed that there may be several more histone gene pairs. The two complete histone H4 genes were different on the nucleotide level but encoded the same amino acid sequence. Comparison of the amino acid sequences of theT. vaginalis H3 and H4 histones with sequences from animals, fungi, and plants as well as other protists revealed a significant divergence not only from the sequences in multicellular organisms but especially from the sequences in other protists likeEntamoeba histolytica, Trypanosoma cruzi, andLeishmania infantum.     

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.     

Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase fromStreptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts

Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During the phase of pentalenolactone production,S. arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed. The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced. Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame. No expression of pentalenolactone-insensitive GAPDH was detected inStreptomyces lividans transformed with the gene. InEscherichia coli, the gene was expressed from an inducedlac promoter. Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH fromS. arenae. Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria. Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding. The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species. The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.Abbreviations GAP Glyceraldehyde-3-phosphate - GAPDH Glyceraldehyde-3-phosphate dehydrogenase     

Nucleotide sequence and molecular evolution of the gene coding for glyceraldehyde-3-phosphate dehydrogenase in the thermoacidophilic archaebacteriumSulfolobus solfataricus

ASulfolobus solfataricus genomic library cloned in the EMBL3 phage was screened using as probes synthetic oligonucleotides designed from the known amino acid sequence of a peptide obtained from the purified glyceraldehyde-3-phosphate dehydrogenase (aGAPD) protein. The screening led to the isolation of six recombinant phages (G1–G6) and one of them (G4) contained the entire GAPD gene. The deduced amino acid sequence accounts for a protein made of 341 amino acids and the initial methionine is encoded by a GTG triplet. Alignment of theS. solfataricus aGAPD sequence versus GAPD from archaea, eukarya, and bacteria showed that aGAPD is very similar to other archaebacterial but not to eukaryotic or eubacterial GAPD. For known archaebacterial GAPD sequences, the rate of nucleotide substitutions per site per year showed that these sequences are homologous not only at the amino acid but also at the nucleotide level. The evolutionary rates are nearly similar to those reported for other eukaryotic genes.This work was supported by grants from the CNR, Target Project on Biotechnology and Bioinstrumentation, and MURST (Rome).     

Glyceraldehyde-3-phosphate dehydrogenase gene diversity in eubacteria and eukaryotes: evidence for intra- and inter-kingdom gene transfer.

Cyanobacteria contain up to three highly divergent glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes: gap1, gap2, and gap3. Genes gap1 and gap2 are closely related at the sequence level to the nuclear genes encoding cytosolic and chloroplast GAPDH of higher plants and have recently been shown to play distinct key roles in catabolic and anabolic carbon flow, respectively, of the unicellular cyanobacterium Synechocystis sp. PCC6803. In the present study, sequences of 10 GAPDH genes distributed across the cyanobacteria Prochloron didemni, Gloeobacter violaceus PCC7421, and Synechococcus PCC7942 and the alpha-proteobacterium Paracoccus denitrificans and the beta-proteobacterium Ralstonia solanacearum were determined. Prochloron didemni possesses homologs to the gap2 and gap3 genes from Anabaena, Gloeobacter harbors gap1 and gap2 homologs, and Synechococcus possesses gap1, gap2, and gap3. Paracoccus harbors two highly divergent gap genes that are related to gap3, and Ralstonia possesses a homolog of the gap1 gene. Phylogenetic analyses of these sequences in the context of other eubacterial and eukaryotic GAPDH genes reveal that divergence across eubacterial gap1, and gap2, and gap3 genes is greater than that between eubacterial gap1 and eukaroytic glycolytic GapC or between eubacterial gap2 and eukaryotic Calvin cycle GapAB. These data strongly support previous analyses which suggested that eukaryotes acquired their nuclear genes for GapC and GapAB via endosymbiotic gene transfer from the antecedents of mitochondria and chloroplasts, and extend the known range of sequence diversity of the antecedent eubacterial genes. Analyses of available GAPDH sequences from other eubacterial sources indicate that the glycosomal gap gene from trypanosomes (cytosolic in Euglena) and the gap gene from the spirochete Treponema pallidum are each other's closest relatives. This specific relationship can therefore not reflect organismal evolution but must be the result of an interkingdom gene transfer, the direction of which cannot be determined with certainty at present. Contrary to this, the origin of the cytosolic Gap gene from trypanosomes can now be clearly defined as gamma-proteobacterial, since the newly established Ralstonia sequence (beta-proteobacteria) branches basally to the gamma-proteobacterial/trypanosomal assemblage.     

Secreted glyceraldehyde-3-phosphate dehydrogenase as a broad spectrum vaccine candidate against microbial infection in aquaculture

Aims: Subcellullar localizations and cross‐immunities of GAPDHs from six common pathogenic bacteria in aquaculture were investigated. Methods and Results: Subcellullar localizations of GAPDHs of Edwardsiella tarda EIB202, Edwardsiella ictaluri ATCC33202, Aeromonas hydrophila LSA34, Vibrio anguillarum MVM425, Vibrio alginolyticus EPGS020401 and Vibrio harveyi VIB647 were analysed with Western blotting, indirect immunofluorescence and flow cytometry examinations. Immunoprotections of different recombinant GAPDHs against these pathogens were investigated with zebrafish model. Western blotting of subcellular extractions showed that all GAPDHs were secreted into extracellular medium and periplasmic space. In addition, GAPDHs were demonstrated to distribute in the outer membranes except MVM425 and VIB647. And, GAPDHs were confirmed to be present on the surface of these bacteria with indirect immunofluorescence and flow cytometry examinations. The remarkable cross‐protective immunities of these recombinant GAPDHs were induced in zebrafish, and the relative protective survivals were almost over 60%. Conclusions: Localizations of GAPDHs from these pathogenic bacteria were similar to many other causative agents. And, GAPDHs could be important protective antigens and give remarkable cross‐immunity against different pathogens. Significance and Impact of the Study: Recombinant GAPDH could be designed as a broad spectrum vaccine candidate against multiple microbial infections in aquaculture.     

Cloning and nucleotide sequence of an archaebacterial glutamine synthetase gene: Phylogenetic implications

Summary The glnA gene of the thermophilic sulphur-dependent archaebacterium Sulfolobus solfataricus was identified by hybridization with the corresponding gene of the cyanobacterium Spirulina platensis and cloned in Escherichia coli. The nucleotide sequence of the 1696 bp DNA fragment containing the structural gene for glutamine synthetase was determined, and the derived amino acid sequence (471 residues) was compared to the sequences of glutamine synthetases from eubacteria and eukaryotes. The homology between the archaebacterial and the eubacterial enzymes is higher (42%–49%) than that found with the eukaryotic counterpart (less than 20%). This was true also when the five most conserved regions, which it is possible to identify in both eubacterial and eukaryotic glutamine synthetases, were analysed.     

Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases

We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N. tabacum. Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago. However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages. Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes. These results strongly support the symbiotic origin of chloroplasts.     

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: An evolutionary scenario of subcellular compartmentation in Kinetoplastida

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72–77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD⁺-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosome, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) - HK hexokinase (EC 2.7.1.1) - PGI glucosephosphate isomerase (EC 5.3.1.9) - PGK phosphoglycerate kinase (EC 2.7.2.3) - PYK pyruvate kinase (EC 2.7.1.40) - TIM triosephosphate isomerase (EC 5.3.1.1) - SDS sodium dodecyl sulfate - SSC saline sodium citrate (0.15 M NaCl, 15 mM sodium citrate, pH 7.0) - MYR millions of years Nucleotide sequence data reported in this paper have been submitted to the EMBL/Genbank/DDBJ nucleotide sequence databases under accession number X74535 Correspondence to: P.A.M. Michels     

Molecular phylogenies based on ribosomal protein L11, L1, L10, and L12 sequences

Summary Available sequences that correspond to the E. coli ribosomal proteins L11, L1, L10, and L12 from eubacteria, archaebacteria, and eukaryotes have been aligned. The alignments were analyzed qualitatively for shared structural features and for conservation of deletions or insertions. The alignments were further subjected to quantitative phylogenetic analysis, and the amino acid identity between selected pairs of sequences was calculated. In general, eubacteria, archaebacteria, and eukaryotes each form coherent and well-resolved nonoverlapping phylogenetic domains. The degree of diversity of the four proteins between the three groups is not uniform. For L11, the eubacterial and archaebacterial proteins are very similar whereas the eukaryotic L11 is clearly less similar. In contrast, in the case of the L12 proteins and to a lesser extent the L10 proteins, the archaebacterial and eukaryotic proteins are similar whereas the eubacterial proteins are different. The eukaryotic L1 equivalent protein has yet to be identified. If the root of the universal tree is near or within the eubacterial domain, our ribosomal protein-based phylogenies indicate that archaebacteria are monophyletic. The eukaryotic lineage appears to originate either near or within the archaebacterial domain. Correspondence to: P. Dennis