Elena Hilario

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.

Inteins, introns, and homing endonucleases: recent revelations about the life cycle of parasitic genetic elements.

Self splicing introns and inteins that rely on a homing endonuclease for propagation are parasitic genetic elements. Their life-cycle and evolutionary fate has been described through the homing cycle. According to this model the homing endonuclease is selected for function only during the spreading phase of the parasite. This phase ends when the parasitic element is fixed in the population. Upon fixation the homing endonuclease is no longer under selection, and its activity is lost through random processes. Recent analyses of these parasitic elements with functional homing endonucleases suggest that this model in its most simple form is not always applicable. Apparently, functioning homing endonuclease can persist over long evolutionary times in populations and species that are thought to be asexual or nearly asexual. Here we review these recent findings and discuss their implications. Reasons for the long-term persistence of a functional homing endonuclease include: More recombination (sexual and as a result of gene transfer) than previously assumed for these organisms; complex population structures that prevent the element from being fixed; a balance between active spreading of the homing endonuclease and a decrease in fitness caused by the parasite in the host organism; or a function of the homing endonuclease that increases the fitness of the host organism and results in purifying selection for the homing endonuclease activity, even after fixation in a local population. In the future, more detailed studies of the population dynamics of the activity and regulation of homing endonucleases are needed to decide between these possibilities, and to determine their relative contributions to the long term survival of parasitic genes within a population. Two outstanding publications on the amoeba Naegleria group I intron (Wikmark et al. BMC Evol Biol 2006, 6: 39) and the PRP8 inteins in ascomycetes (Butler et al.BMC Evol Biol 2006, 6: 42) provide important stepping stones towards integrated studies on how these parasitic elements evolve through time together with, or despite, their hosts.

The Prokaryote-to-Eukaryote Transition Reflected in the Evolution of the V/F/A-ATPase Catalytic and Proteolipid Subunits

Abstract. 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.

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 Earths 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.

Gene duplications and horizontal gene transfer during early evolution

The evolutionary history of organisms can be reconstructed using various information sources: the fossil and geological records, the comparative analysis of biochemical pathways, and the reconstruction of molecular phylogenies from macromolecules found in extant organisms. In the absence of a complete archaean microfossil record and unique morphological characters for most microbial groups, molecular markers have been used to unravel the relationships between the major groups ofprokaryotes. The best studied of these molecules is undoubtedly the small subunit ribosomal RNA, but many other macromolecules, especially protein sequences have been useful in studying the relationships among prokaryotic kingdoms and domains. However, at best, the analysis of extant macromolecules can only yield information on tile phylogeny of the molecules under study. Even if molecular phylogenies could be resolved without ambiguity, the step from molecular phylogeny to species phylogeny would remain complicated because genetic information has also been transferred horizontally between independent evolutionary lineages. It is therefore not surprising that comparative analysis of different molecular phylogenies reveals a net-like structure of the species phylogeny (Gogarten, 1995; Hiliario & Gogarten, 1993). Differences between well resolved molecular phylogenies can be due either to unrecognized gene duplications or to horizontal gene transfer. Two examples that conflict with well documented phylogenetic relationships are the presence of archaeal type H+-ATPases in Thermus and Enterococcus and the close association between Gram positive eubacteria and archaea as supported by many different genes, including heat shock proteins (HSP70), ghitamine synthetases and glutamate dehydrogenases. In both cases the best explanation for these conflicts is horizontal gene transfer. Assuming unrecognized paralogies (duplicated genes) as an explanation would necessitate many instances of convergent evolution; furthermore this assumption would also result in species phylogenies that are at odds with two distinct prokaryotic domains (archaea and eubacteria). The large number of characters that reflect the close association between archaea and eubacteria suggest that a substantial portion of the eubacterial genome participated in this transfer. Horizontal gene transfer as a possible evolutionary mechanism gives as a result net-like species phylogenies that complicate inferring the properties of the last common ancestor. Even so, the data strongly indicate that the last common ancestor was a cellular organism, with a DNA based genome, and a sophisticated transcription and translation machinery. Furthermore, the analysis of ATPase structure function relationships and the evolution of cytochrome oxidase homologues suggest that the last

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

BackgroundInteins 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.ResultsThe 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.ConclusionsDespite 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.

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.

End Labeling Procedures

There are two ways to label a DNA molecular; by the ends or all along the molecule. End labeling can be performed at the 3′- or 5′-end. Labeling at the 3′ end is performed by filling 3′-end recessed ends with a mixture or labeled and unlabeled dNTPs using Klenow or T4 DNA polymerases. Both reactions are template dependent. Terminal deoxynucleotide transferase incorporates dNTPs at the 3′ end of any kind of DNA molecule or RNA. Labels incorporated at the 3′-end of the DNA molecule prevent any further extension or ligation to any other molecule, but this can be overcome by labeling the 5′-end of the desired DNA molecule. 5′-end labeling is performed by enzymatic methods (T4 polynucleotide kinase exchange and forward reactions), by chemical modification of sensitized oligonucleotides with phosphoroamidite, or by combined methods. Probe cleanup is recommended when high background problems occur, but caution should be taken not to damage the attached probe with harsh chemicals or by light exposure.

Overview of hybridization and detection techniques.

A misconception regarding the sensitivity of nonradioactive methods for screening genomic DNA libraries often hinders the establishment of these environmentally friendly techniques in molecular biology laboratories. Nonradioactive probes, properly prepared and quantified, can detect DNA target molecules to the femtomole range. However, appropriate hybridization techniques and detection methods should also be adopted for an efficient use of nonradioactive techniques. Detailed descriptions of genomic library handling before and during the nonradioactive hybridization and detection are often omitted from publications. This chapter aims to fill this void by providing a collection of technical tips on hybridization and detection techniques.