Orthologs, paralogs and genome comparisons.

During the past decade, ancient gene duplications were recognized as one of the main forces in the generation of diverse gene families and the creation of new functional capabilities. New tools developed to search data banks for homologous sequences, and an increased availability of reliable three-dimensional structural information led to the recognition that proteins with diverse functions can belong to the same superfamily. Analyses of the evolution of these superfamilies promises to provide insights into early evolution but are complicated by several important evolutionary processes. Horizontal transfer of genes can lead to a vertical spread of innovations among organisms, therefore finding a certain property in some descendants of an ancestor does not guarantee that it was present in that ancestor. Complete or partial gene conversion between duplicated genes can yield phylogenetic trees with several, apparently independent gene duplications, suggesting an often surprising parallelism in the evolution of independent lineages. Additionally, the breakup of domains within a protein and the fusion of domains into multifunctional proteins makes the delineation of superfamilies a task that remains difficult to automate.

Unsupervised learning in detection of gene transfer.

The tree representation as a model for organismal evolution has been in use since before Darwin. However, with the recent unprecedented access to biomolecular data, it has been discovered that, especially in the microbial world, individual genes making up the genome of an organism give rise to different and sometimes conflicting evolutionary tree topologies. This discovery calls into question the notion of a single evolutionary tree for an organism and gives rise to the notion of an evolutionary consensus tree based on the evolutionary patterns of the majority of genes in a genome embedded in a network of gene histories. Here, we discuss an approach to the analysis of genomic data of multiple genomes using bipartition spectral analysis and unsupervised learning. An interesting observation is that genes within genomes that have evolutionary tree topologies, which are in substantial conflict with the evolutionary consensus tree of an organism, point to possible horizontal gene transfer events which often delineate significant evolutionary events.

The V-ATPase A subunit gene (vma-1) from Giardia lamblia.

The sequence of the gene encoding the A subunit of the vacuolar type ATPase from Giardia lamblia is reported. Comparison of the encoded protein with the homologous subunits of eukaryotic and archaebacterial ATPases reveals high levels of similarity throughout the sequence (e.g., overall 49.1 and 44.6% identity to the homologous subunit from carrot and Halobacterium, respectively). An exception are three regions which are unique to the Giardia subunit. The largest of these regions contains motifs characteristic for eukaryotic spliceosomal introns; however, comparison to the cDNA shows that this region is also present in the mRNA.

A conserved intron in the V-ATPase A subunit genes of plants and algae.

Amplification and sequencing of part of the coding regions of the catalytic V-type ATPase subunit shows the presence of (at least) two genes in all land plants as well as the conservative insertion of a noncoding sequence. The two genes exhibit a coding region of the same length but differ in the number of nucleotides present in the intron. The latter is surprisingly conserved suggesting the presence of functional constraints on the intron sequences. The findings presented in this report indicate that introns from plants and animals are characterized by different internal structural elements.

The H+ ATPase regulatory subunit of Methanococcus thermolithotrophicus: amplification of an 800 bp fragment by polymerase chain reaction.

An 800 bp fragment of Methanococcus thermolithotrophicus genomic DNA was amplified by the polymerase chain reaction method using primers designed from conserved regions of the V-type H+ ATPase regulatory subunits from the archaebacterium Sulfolobus, and several eukaryotes. Although more than one product was obtained, only one of them had the expected size and was exclusively amplified in the presence of the left and right primers. The DNA and the deduced protein sequences of the putative Methanococcus H+ ATPase subunit revealed homology to the corresponding sequences in Sulfolobus and eukaryotes (about 60% identical residues) and a less evident homology to the eubacterial F1-ATPase alpha-subunit (22% identical residues with E. coli).

The intein of the Thermoplasma A-ATPase A subunit: structure, evolution and expression in E. coli.

BACKGROUND: Inteins are selfish genetic elements that excise themselves from the host protein during post translational processing, and religate the host protein with a peptide bond. In addition to this splicing activity, most reported inteins also contain an endonuclease domain that is important in intein propagation. RESULTS: The gene encoding the Thermoplasma acidophilum A-ATPase catalytic subunit A is the only one in the entire T. acidophilum genome that has been identified to contain an intein. This intein is inserted in the same position as the inteins found in the ATPase A-subunits encoding gene in Pyrococcus abyssi, P. furiosus and P. horikoshii and is found 20 amino acids upstream of the intein in the homologous vma-1 gene in Saccharomyces cerevisiae. In contrast to the other inteins in catalytic ATPase subunits, the T. acidophilum intein does not contain an endonuclease domain.T. acidophilum has different codon usage frequencies as compared to Escherichia coli. Initially, the low abundance of rare tRNAs prevented expression of the T. acidophilum A-ATPase A subunit in E. coli. Using a strain of E. coli that expresses additional tRNAs for rare codons, the T. acidophilum A-ATPase A subunit was successfully expressed in E. coli. CONCLUSIONS: Despite differences in pH and temperature between the E. coli and the T. acidophilum cytoplasms, the T. acidophilum intein retains efficient self-splicing activity when expressed in E. coli. The small intein in the Thermoplasma A-ATPase is closely related to the endonuclease containing intein in the Pyrococcus A-ATPase. Phylogenetic analyses suggest that this intein was horizontally transferred between Pyrococcus and Thermoplasma, and that the small intein has persisted in Thermoplasma apparently without homing.

Kinetics and Specificity of ATP-dependent Proton Translocation Measured with Acridine Orange in Microsomal Fractions from Green Suspension Cells of Chenopodium rubrum L.

From photoautotrophic cell suspension cultures of Chenopodium rubrum L. microsomal fractions were prepared by differential and isopycnic sucrose gradient centrifugation. ATP-generated proton-accumulation was studied on a microsomal fraction (MF) of proton-translocating vesicles with a density of 1.11 to 1.12 gem(-3). This activity did not overlap with mitochondrial or chloroplast markers. MF is presumably of tonoplast origin. The assay was based upon pH-dependent acridine orange (AO) absorption shifts at 490 nm, Δ(490), due to AO-accumulation and polymerization within the vesicles, and was calibrated using pK and K(D) for AO polymers given by Zanker (Z. physikal. Chemie 199, 225, 1952). Addition of 1 mM ATP to MF yields a transmembrane ΔpH of about 2 units within 10 min (kinetics with a single time constant, τ=400s) and collapses after addition of the protonophor CCCP (τ=22s). The proton pumping activity is insensitive to vanadate and highly specific for ATP compared with GTP, UTP, CTP, and ITP, and half-saturates at 0.38 to 0.46 mM ATP, indicated by both initial rate (1 min) and steady state value of Δ(490) (10 min). Proton pumping into and proton leakage from the vesicles was cast into a feedback-scheme showing that the pump is working at a constant rate, that is, independently of the size of the created ΔpH.

Horizontal transfer of ATPase genes--the tree of life becomes a net of life.

An ancient gene duplication gave rise to the catalytic and non-catalytic subunits of each of the three types of proton pumping ATPases: vacuolar, archaebacterial and eubacterial. Previously, this gene duplication has been used to root the universal tree of life. However, recent findings of archaebacterial type ATPases in eubacteria and of eubacterial type in an archaebacterium suggested that both types of ATPases may have been already present in the last common ancestor. Here we show that a phylogenetic analysis of these ATPase subunits indicates that this conclusion is premature. We suggest that horizontal gene transfer can explain the data. In addition, we show that the analysis of glutamate dehydrogenases data neither affirm nor contradict any particular placement of the last common ancestor in the universal tree of life. The prevalence and the mode of horizontal gene transfer is discussed.

Two separate genes encode the catalytic 70 kDa V-ATPase subunit in Psilotum and Equisetum.

Vacuolar type ATPases have been found on various endomembranes of eukaryotic cells, e.g. lysosomes, chromaffin granules, vesicles derived from the Golgi apparatus, endosomes and vacuoles. Although this ATPase type is targeted to different compartments in one cell, only one gene for each subunit had been found per genome. Using PCR across intron-exon boundaries we show that two different genes encode the catalytic subunit of the V-ATPase in Psilotum nudum and Equisetum arvense. The substitution rates for the three codon positions and the intervening sequences show that in Psilotum both genes are transcribed and are under selection pressure, however, this seems not to be the case for Equisetum. The relatively high similarity between the two genes found in each species as compared to the interspecies similarities suggest that for some time after the gene duplication had occurred the two copies were subject to gene conversion mechanisms. An unexpected degree of conservation of the intervening sequences themselves is noted and statistically verified, however, no structural constraints that could explain these findings were detected.

Molecular evolution of H+-ATPases. I. Methanococcus and Sulfolobus are monophyletic with respect to eukaryotes and Eubacteria.

The classification of methanogenic bacteria as archaebacteria based on 16 s rRNA sequence analysis is currently in dispute. To provide an alternative molecular marker, the polymerase chain reaction technique was used to amplify a 930 bp fragment of Methanococcus thermolithotrophicus genomic DNA corresponding to the catalytic domain of the membrane H+-ATPase. The deduced amino acid sequence was 54-58% identical to the approximately 70 kDa subunits of Sulfolobus acidocaldarius and the eukaryotic vacuolar-type H+-ATPase, and only 29% identical to the beta subunit of the eubacterial-type F0F1-ATPases. Interestingly, a highly conserved aspartate residue in the phosphorylation domain of E1E2-ATPases (P-type) is conserved in the Methanococcus sequence, but is absent from all other known vacuolar and F0F1-ATPases. This suggests that the H+-ATPase of M. thermolithotrophicus, like that of M. voltae, may have a phosphorylated intermediate, despite belonging to the vacuolar-type class of proton pumps. Phylogenetic analysis using Felsenstein's maximum likelihood method and Lake's evolutionary parsimony method confirmed that the H+-ATPases of the two archaebacteria, Methanococcus and Sulfolobus, when compared to eukaryotic vacuolar-type ATPases and eubacterial F0F1-ATPases, form a monophyletic group.

The early evolution of cellular life.

Recent progress in data collection and analysis has changed the study of origin of life from an area dominated by speculation into a field abundant with testable hypotheses. This review discusses advances in the following areas: the fossil recordsd; the 'retrodiction' of biochemical pathways; and contradictions between different molecular phylogenies. The latter indicates a limited number of horizontal gene transfers during the early evolution. However, these cases of horizontal gene transfer are so infrequent that they can be detected as exceptions in an otherwise coherent picture. Cases of horizontal gene transfer can be recognized within the background of the majority consensus of molecular markers. The fusion of separate lineages to form new species is revealed by the simultaneous horizontal transfer of several independent genes.

Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.

Evolution of proton pumping ATPases: Rooting the tree of life.

Proton pumping ATPases are found in all groups of present day organisms. The F-ATPases of eubacteria, mitochondria and chloroplasts also function as ATP synthases, i.e., they catalyze the final step that transforms the energy available from reduction/oxidation reactions (e.g., in photosynthesis) into ATP, the usual energy currency of modern cells. The primary structure of these ATPases/ATP synthases was found to be much more conserved between different groups of bacteria than other parts of the photosynthetic machinery, e.g., reaction center proteins and redox carrier complexes.These F-ATPases and the vacuolar type ATPase, which is found on many of the endomembranes of eukaryotic cells, were shown to be homologous to each other; i.e., these two groups of ATPases evolved from the same enzyme present in the common ancestor. (The term eubacteria is used here to denote the phylogenetic group containing all bacteria except the archaebacteria.) Sequences obtained for the plasmamembrane ATPase of various archaebacteria revealed that this ATPase is much more similar to the eukaryotic than to the eubacterial counterpart. The eukaryotic cell of higher organisms evolved from a symbiosis between eubacteria (that evolved into mitochondria and chloroplasts) and a host organism. Using the vacuolar type ATPase as a molecular marker for the cytoplasmic component of the eukaryotic cell reveals that this host organism was a close relative of the archaebacteria.A unique feature of the evolution of the ATPases is the presence of a non-catalytic subunit that is paralogous to the catalytic subunit, i.e., the two types of subunits evolved from a common ancestral gene. Since the gene duplication that gave rise to these two types of subunits had already occurred in the last common ancestor of all living organisms, this non-catalytic subunit can be used to root the tree of life by means of an outgroup; that is, the location of the last common ancestor of the major domains of living organisms (archaebacteria, eubacteria and eukaryotes) can be located in the tree of life without assuming constant or equal rates of change in the different branches.A correlation between structure and function of ATPases has been established for present day organisms. Implications resulting from this correlation for biochemical pathways, especially photosynthesis, that were operative in the last common ancestor and preceding life forms are discussed.

The prokaryote-to-eukaryote transition reflected in the evolution of the V/F/A-ATPase catalytic and proteolipid subunits.

Changes in the primary and quarternary structure of vacuolar and archaeal type ATPases that accompany the prokaryote-to-eukaryote transition are analyzed. The gene encoding the vacuolar-type proteolipid of the V-ATPase from Giardia lamblia is reported. Giardia has a typical vacuolar ATPase as observed from the common motifs shared between its proteolipid subunit and other eukaryotic vacuolar ATPases, suggesting that the former enzyme works as a hydrolase in this primitive eukaryote. The phylogenetic analyses of the V-ATPase catalytic subunit and the front and back halves of the proteolipid subunit placed Giardia as the deepest branch within the eukaryotes. Our phylogenetic analysis indicated that at least two independent duplication and fusion events gave rise to the larger proteolipid type found in eukaryotes and in Methanococcus. The spatial distribution of the conserved residues among the vacuolar-type proteolipids suggest a zipper-type interaction among the transmembrane helices and surrounding subunits of the V-ATPase complex. Important residues involved in the function of the F-ATP synthase proteolipid have been replaced during evolution in the V-proteolipid, but in some cases retained in the archaeal A-ATPase. Their possible implication in the evolution of V/F/A-ATPases is discussed.

Substrate specifity of the hexose carrier in the plasmalemma of Chenopodium suspension cells probed by transmembrane exchange diffusion.

Substrate specifity of the proton-driven hexose cotransport carrier in the plasmalemma of photoautotrophic suspension cells of Chenopodium rubrum L. has been studies through the short-term perturbation of (14)C-labelled efflux of 3-O-methyl-D-glucose. Efflux, occurring exclusively via carrier-mediated exchange diffusion, is trans-stimulated by the substrate and trans-inhibited by the glucose-transport inhibitors phlorizin (K 1/2=7.9 mM) and its aglucon phloretin (K 1/2=84 µM); with both inhibitors, 3-O-methyl-D-glucose efflux may be blocked completely. Trans-stimulation of efflux (up to fourfold) by a variety of the D-enantiomers of neutral hexoses, including glucose (K 1/2=48 µM), 3-O-methyl-D-glucose (K 1/2=139 µM), and fructose (K 1/2=730 µM), but not by, for instance, D-allose, and L-sorbose, shows that carrier-substrate interaction critically involves the axial position at C-1 and C-3, respectively. We suggest that substrate binding by the Chenopodium hexose carrier involves both hydrophobic interaction with the pyran-ring and hydrogen-ion bonding at C-1 and C-3 of the D-glucose conformation.

The effects of heavy meteorite bombardment on the early evolution--the emergence of the three domains of life.

A characteristic of many molecular phylogenies is that the three domains of life (Bacteria, Archaea, Eucarya) are clearly separated from each other. The analyses of ancient duplicated genes suggest that the last common ancestor of all presently known life forms already had been a sophisticated cellular prokaryote. These findings are in conflict with theories that have been proposed to explain the absence of deep branching lineages. In this paper we propose an alternative scenario, namely, a large meteorite impact that wiped out almost all life forms present on the early Earth. Following this nearly complete frustation of life on Earth, two surviving extreme thermophilic species gave rise to the now existing major groups of living organisms, the Bacteria and Archaea. [The latter also contributed the major portion to the nucleo-cytoplasmic component of the Eucarya]. An exact calibration of the molecular record with regard to time is not yet possible. The emergence of Eucarya in fossil and molecular records suggests that the proposed late impact should have occurred before 2100 million years before present (BP). If the 3500 million year old microfossils [Schopf, J. W. 1993: Science 260: 640-646] are interpreted as representatives of present day existing groups of bacteria (i.e., as cyanobacteria), then the impact is dated to around 3700 million years BP. The analysis of molecular sequences suggests that the separation between the Eucarya and the two prokaryotic domains is less deep then the separation between Bacteria and Archaea. The fundamental cell biological differences between Archaea and Eucarya were obtained over a comparatively short evolutionary distance (as measured in number of substitution events in biological macromolecules). Our interpretation of the molecular record suggests that life emerged early in Earth's history even before the time of the heavy bombardment was over. Early life forms already had colonized extreme habitats which allowed at least two prokaryotic species to survive a late nearly ocean boiling impact. The distribution of ecotypes on the rooted universal tree of life should not be interpreted as evidence that life originated in extremely hot environments.