Molecular Parasites That Evolve Longer Genomes

Molecular parasites that utilize the replication machinery of cells or of in vitro amplification reactions have previously been characterized. By and large, these parasites have been smaller than the viruses or amplicons that gave rise to them. This is likely because shorter genomes can be replicated more quickly. In contrast, we have identified and characterized parasites of an isothermal amplification reaction that are longer than their parental molecules yet replicate much more efficiently. These results raise interesting questions regarding whether the optimal size of replicators reflects a trade-off between the information encoded in a parasite and the information encoded in the machinery replicating that parasite. Received: 9 February 1999 / Accepted: 7 June 1999     

In Vitro Culture of Rudimentary Embryos of Ilex paraguariensis: Responses to Exogenous Cytokinins

The effects of benzyladenine (BAP), kinetin (KIN), zeatin (ZEA), isopentenyladenine (2iP), and thidiazuron (TDZ) were studied on in vitro growth of rudimentary embryos of Ilex paraguariensis St. Hil. Heart stage zygotic embryos were removed from seeds of immature, light green fruits and cultured aseptically on quarter-strength Murashige and Skoog medium containing 3% sucrose, 0.65% agar, and supplemented with or without three concentrations of BAP, KIN, ZEA, 2iP, or TDZ. Cultures were incubated in darkness at 27 ± 2°C. Media containing 4.4 × 10⁻⁶ m BAP, 4.6 × 10⁻⁶ m KIN, or 4.9 × 10⁻⁶ m 2iP were totally ineffective in inducing embryo growth after culture for 28 days. However, lower concentrations of these compounds (4.4 × 10⁻⁸ m BAP, 4.6 × 10⁻⁸ m KIN, 4.5 × 10⁻⁸ m ZEA, or 4.9 × 10⁻⁸ m 2iP) promoted embryo growth. TDZ at 9.9 × 10⁻⁹ m, 9.9 × 10⁻⁸ m, or 9.9 × 10⁻⁷ m induced embryo growth at similar rates. The maximum percentage of embryos converted to seedlings was achieved when the medium was supplemented with 4.5 × 10⁻⁷ m ZEA. Received August 1, 1997; accepted February 19, 1998     

Fast Evolution of Interleukin-2 in Mammals and Positive Selection in Ruminants

Interleukin-2 (IL-2) is a cytokine involved in induction and regulation of the immune response in mammals. There have been numerous reports about the search for IL-2 in species other than mammals, and recently an IL-2-like gene has been isolated in chicken. Using PCR, we searched for IL-2 gene sequences in a wide variety of mammals, including marsupials and monotremes, as well as in birds. Although we can readily amplify IL-2 gene fragments in placental mammals, no amplification was obtained in other species. This is best explained by very high substitution rates. This suggest that strategies to isolate IL-2 homologous genes outside mammals should involve functional assays, as for the chicken gene, and not hybridization-based techniques. Nonsynonymous substitution rates are especially high in ruminants, due to positive selection acting on regions important in term of structure-function. We suggest that, although globally similar, the immune response of various mammals is not identical, mainly at the level of cytokine-mediated regulations. Received: 27 July 1999 / Accepted: 15 April 2000     

Evaluation of Sequence Variation and Selection in the Bindin Locus of the Red Sea Urchin, Strongylocentrotus franciscanus

Recent evidence suggests that gamete recognition proteins may be subjected to directed evolutionary pressure that enhances sequence variability. We evaluated whether diversity enhancing selection is operating on a marine invertebrate fertilization protein by examining the intraspecific DNA sequence variation of a 273-base pair region located at the 5′ end of the sperm bindin locus in 134 adult red sea urchins (Strongylocentrotus franciscanus). Bindin is a sperm recognition protein that mediates species-specific gamete interactions in sea urchins. The region of the bindin locus examined was found to be polymorphic with 14 alleles. Mean pairwise comparison of the 14 alleles indicates moderate sequence diversity (p-distance = 1.06). No evidence of diversity enhancing selection was found. It was not possible to reject the null hypothesis that the sequence variation observed in S. franciscanus bindin is a result of neutral evolution. Statistical evaluation of expected proportions of replacement and silent nucleotide substitutions, observed versus expected proportions of radical replacement substitutions, and conformance to the McDonald and Kreitman test of neutral evolution all indicate that random mutation followed by genetic drift created the polymorphisms observed in bindin. Observed frequencies were also highly similar to results expected for a neutrally evolving locus, suggesting that the polymorphism observed in the 5′ region of S. franciscanus bindin is a result of neutral evolution. Received: 19 June 1998 / Accepted: 2 August 2000     

The Role of Interelement Selection in Saccharomyces cerevisiae Ty Element Evolution

Retrotransposons are mobile genetic elements that are ubiquitous components of eukaryotic genomes. The evolutionary success of retrotransposons is explained by their ability to replicate faster than the host genomes in which they reside. Elements with higher rates of genomic replication possess a selective advantage over less active elements. Retrotransposon populations, therefore, are shaped largely by selective forces acting at the genomic level between elements. To evaluate rigorously the effects of selective forces acting on retrotransposons, detailed information on the patterns of molecular variation within and between retrotransposon families is needed. The sequencing of the Saccharomyces cerevisiae genome, which includes the entire genomic complement of yeast retrotransposons, provides an unprecedented opportunity to access and analyze such data. In this study, we analyzed in detail the patterns of nucleotide variation within the open reading frames of two parental (Ty1 and Ty2) and one hybrid (Ty1/2) family of yeast retrotransposons. The pattern and distribution of nucleotide changes on the phylogenetic reconstructions of the three families of Ty elements reveal evidence of negative selection on both internal and external branches of the Ty phylogenies. These results indicate that most, if not all, Ty elements examined represent active or recently active retrotransposon lineages. We discuss the relevance of these findings with respect to the coevolutionary dynamic operating between genomic element populations and the host organisms in which they reside. Received: 5 November 1998 / Accepted: 17 March 1999     

Variable Immune-Driven Natural Selection in the Attachment (G) Glycoprotein of Respiratory Syncytial Virus (RSV)

A maximum-likelihood analysis of selection pressures acting on the attachment (G) glycoprotein gene of respiratory syncytial virus (RSV) from humans (HRSV) and bovines (BRSV) is presented. Six positively selected sites were identified in both group A and group B of HRSV, although only one site was common between them, while no positively selected sites were detected in BRSV. All positively selected sites were located within the ectodomain of the G protein and showed some association with positions of immunoglobulin (Ig) epitopes and sites of O-glycosylation. These results suggest that immune (antibody)-driven natural selection is an important determinant of RSV evolution and that this selection pressure differs among strains. The passage histories of RSV strains were also shown to affect the distribution of positively selected sites, particularly in HRSV B, and should be considered whenever retrospective analysis of adaptive evolution is undertaken. Received: 15 August 2000 / Accepted: 2 November 2000     

Phylogenetic Reconstruction Using an Unsupervised Growing Neural Network That Adopts the Topology of a Phylogenetic Tree

We propose a new type of unsupervised, growing, self-organizing neural network that expands itself by following the taxonomic relationships that exist among the sequences being classified. The binary tree topology of this neutral network, contrary to other more classical neural network topologies, permits an efficient classification of sequences. The growing nature of this procedure allows to stop it at the desired taxonomic level without the necessity of waiting until a complete phylogenetic tree is produced. This novel approach presents a number of other interesting properties, such as a time for convergence which is, approximately, a lineal function of the number of sequences. Computer simulation and a real example show that the algorithm accurately finds the phylogenetic tree that relates the data. All this makes the neural network presented here an excellent tool for phylogenetic analysis of a large number of sequences. Received: 14 May 1996 / Accepted: 6 August 1996     

Accumulation of Species-Specific Amino Acid Replacements That Cause Loss of Particular Protein Functions in Buchnera, an Endocellular Bacterial Symbiont

Endosymbiotic bacteria live in animal cells and are transmitted vertically at the time of the host's reproduction. In view of their small and asexual populations with infrequent chances of recombination, these endocellular bacteria are expected to accumulate mildly deleterious mutations. Previous studies showed that the DNA sequences of these bacteria evolved faster than those of free-living bacteria. In this study, we compared all the ORFs of Buchnera, an endocellular bacterial symbiont of aphids, with those of 34 other prokaryotic organisms and estimated the effect of the accelerated evolution of Buchnera on the functions of its proteins. It was revealed that Buchnera proteins contain many mutations at the sites where sequences are conserved in their orthologues in many other organisms. In addition, amino acid replacements at the conserved sites are mostly changes to physicochemically different amino acids. These results suggest that functions and conformations of Buchnera proteins have been seriously impaired or strongly modified. Indeed, extensive loss of functional motifs was observed in some Buchnera proteins. In many Buchnera proteins mutations were not detected evenly throughout each molecule but tended to accumulate in some functional units, possibly leading to loss of specific functions. As Buchnera has an unusual and limited gene repertory, it is conceivable that the manner of interactions among its proteins has been changed, and thus, functional constraints over their amino acid residues have also been changed during evolution. This may account for the loss of some functional units only in the Buchnera proteins. We obtained evidence that amino acid replacements in Buchnera were not always deleterious, but neutral or, in some cases, even positively selected. Received: 14 December 2000 / Accepted: 12 March 2001     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001     

Selection on the codon bias of chloroplast and cyanelle genes in different plant and algal lineages

In the plant chloroplast genome the codon usage of the highly expressed psbA gene is unique and is adapted to the tRNA population, probably due to selection for translation efficiency. In this study the role of selection on codon usage in each of the fully sequenced chloroplast genomes, in addition to Chlamydomonas reinhardtii, is investigated by measuring adaptation to this pattern of codon usage. A method is developed which tests selection on each gene individually by constructing sequences with the same amino acid composition as the gene and randomly assigning codons based on the nucleotide composition of noncoding regions of that genome. The codon bias of the actual gene is then compared to a distribution of random sequences. The data indicate that within the algae selection is strong in Cyanophora paradoxa, affecting a majority of genes, of intermediate intensity in Odontella sinensis, and weaker in Porphyra purpurea and Euglena gracilis. In the plants, selection is found to be quite weak in Pinus thunbergii and the angiosperms but there is evidence that an intermediate level of selection exists in the liverwort Marchantia polymorpha. The role of selection is then further investigated in two comparative studies. It is shown that average relative codon bias is correlated with expression level and that, despite saturation levels of substitution, there is a strong correlation among the algae genomes in the degree of codon bias of homologous genes. All of these data indicate that selection for translation efficiency plays a significant role in determining the codon bias of chloroplast genes but that it acts with different intensities in different lineages. In general it is stronger in the algae than the higher plants, but within the algae Euglena is found to have several unusual features which are noted. The factors that might be responsible for this variation in intensity among the various genomes are discussed. Received: 6 June 1997 / Accepted: 24 July 1997     

Analysis of Ribosomal RNA Genes Suggests That Trypanosomes Are Monophyletic

To further investigate the phylogeny of protozoa from the order Kinetoplastida we have sequenced the small subunit (SSU) and a portion of the large subunit (LSU) nuclear rRNA genes. The SSU and LSU sequences were determined from a lizard trypanosome, Trypanosoma scelopori and a bodonid, Rhynchobodo sp., and the LSU sequences were determined from an insect trypanosomatid, Crithidia oncopelti, and a bodonid, Dimastigella trypaniformis. Contrary to previous results, in which trypanosomes were found to be paraphyletic, with Trypanosoma brucei representing the earliest-diverging lineage, we have now found evidence for the monophyly of trypanosomes. Addition of new taxa which subdivide long branches (such as that of T. brucei) have helped to identify homoplasies responsible for the paraphyletic trees in previous studies. Although the monophyly of the trypanosome clade is supported in the bootstrap analyses for maximum likelihood at 97% and maximum parsimony at 92%, there is only a small difference in ln-likelihood value or tree length between the most optimal monophyletic tree and the best suboptimal paraphyletic tree. Within the trypanosomatid subtree, the clade of trypanosomes is a sister group to the monophyletic clade of the nontrypanosome genera. Different groups of trypanosomes group on the tree according to their mode of transmission. This suggests that the adaptation to invertebrate vectors plays a more important role in the trypanosome evolution than the adaptation to vertebrate hosts. Received: 5 July 1996 / Accepted: 26 September 1996     

Evolution of Rh Blood Group Genes Have Experienced Gene Conversions and Positive Selection

There are two tightly linked loci (D and CE) for the human Rh blood group. Their gene products are membrane proteins having 12 transmembrane domains and form a complex with Rh50 glycoprotein on erythrocytes. We constructed phylogenetic networks of human and nonhuman primate Rh genes, and the network patterns suggested the occurrences of gene conversions. We therefore used a modified site-by-site reconstruction method by using two assumed gene trees and detected 9 or 11 converted regions. After eliminating the effect of gene conversions, we estimated numbers of nonsynonymous and synonymous substitutions for each branch of both trees. Whichever gene tree we selected the branch connecting hominoids and Old World monkeys showed significantly higher nonsynonymous than synonymous substitutions, an indication of positive selection. Many other branches also showed higher nonsynonymous than synonymous substitutions; this suggests that the Rh genes have experienced some kind of positive selection. Received: 16 March 1999 / Accepted: 17 June 1999     

Positive Darwinian Selection Promotes Heterogeneity Among Members of the Antifreeze Protein Multigene Family

A variety of organisms have independently evolved proteins exhibiting antifreeze activity that allows survival at subfreezing temperatures. The antifreeze proteins (AFPs) bind ice nuclei and depress the freezing point by a noncolligative absorption–inhibition mechanism. Many organisms have a heterogeneous suite of AFPs with variation in primary sequence between paralogous loci. Here, we demonstrate that the diversification of the AFP paralogues is promoted by positive Darwinian selection in two independently evolved AFPs from fish and beetle. First, we demonstrate an elevated rate of nonsynonymous substitutions compared to synonymous substitutions in the mature protein coding region. Second, we perform phylogeny-based tests of selection to demonstrate a subset of codons is subjected to positive selection. When mapped onto the three-dimensional structure of the fish antifreeze type III antifreeze structure, these codons correspond to amino acid positions that surround but do not interrupt the putative ice-binding surface. The selective agent may be related to efficient binding to diverse ice surfaces or some other aspect of AFP function. Received: 27 February 2001 / Accepted: 12 September 2001     

Synonymous Codon Choices in the Extremely GC-Poor Genome of Plasmodium falciparum: Compositional Constraints and Translational Selection

We have analyzed the patterns of synonymous codon preferences of the nuclear genes of Plasmodium falciparum, a unicellular parasite characterized by an extremely GC-poor genome. When all genes are considered, codon usage is strongly biased toward A and T in third codon positions, as expected, but multivariate statistical analysis detects a major trend among genes. At one end genes display codon choices determined mainly by the extreme genome composition of this parasite, and very probably their expression level is low. At the other end a few genes exhibit an increased relative usage of a particular subset of codons, many of which are C-ending. Since the majority of these few genes is putatively highly expressed, we postulate that the increased C-ending codons are translationally optimal. In conclusion, while codon usage of the majority of P. falciparum genes is determined mainly by compositional constraints, a small number of genes exhibit translational selection. Received: 10 November 1998 / Accepted: 28 January 1999     

The Ghost of Selection Past: Rates of Evolution and Functional Divergence of Anciently Duplicated Genes

The duplication of genes and even complete genomes may be a prerequisite for major evolutionary transitions and the origin of evolutionary novelties. However, the evolutionary mechanisms of gene evolution and the origin of novel gene functions after gene duplication have been a subject of many debates. Recently, we compiled 26 groups of orthologous genes, which included one gene from human, mouse, and chicken, one or two genes from the tetraploid Xenopus and two genes from zebrafish. Comparative analysis and mapping data showed that these pairs of zebrafish genes were probably produced during a fish-specific genome duplication that occurred between 300 and 450 Mya, before the teleost radiation (Taylor et al. 2001). As discussed here, many of these retained duplicated genes code for DNA binding proteins. Different models have been developed to explain the retention of duplicated genes and in particular the subfunctionalization model of Force et al. (1999) could explain why so many developmental control genes have been retained. Other models are harder to reconcile with this particular set of duplicated genes. Most genes seem to have been subjected to strong purifying selection, keeping properties such as charge and polarity the same in both duplicates, although some evidence was found for positive Darwinian selection, in particular for Hox genes. However, since only the cumulative pattern of nucleotide substitutions can be studied, clear indications of positive Darwinian selection or neutrality may be hard to find for such anciently duplicated genes. Nevertheless, an increase in evolutionary rate in about half of the duplicated genes seems to suggest that either positive Darwinian selection has occurred or that functional constraints have been relaxed at one point in time during functional divergence. Received: 4 January 2001 / Accepted: 29 March 2001     

SITS Blockade Induces Multiple Subconductance States in a Large Conductance Chloride Channel

The effect of the chloride channel blocker 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid (SITS) on the gating and amplitude of an endothelial chloride channel was explored using the outside-out configuration of the patch-clamp technique. Under control conditions the channel displayed two main gating modes: shut and fully open. Transitions to equally spaced subconductance states were rarely observed (less than 10 events/minute). At low concentrations (<45 μm), SITS increased the number of transitions to the three subconductance states in a concentration-dependent manner, while reducing the number of transitions to the fully open state. This effect was maintained after removing SITS from the bath solution, suggesting that the modifications in the channel induced by SITS were irreversible. All four conducting states had similar current-voltage relationships. At higher concentrations (>45 μm), SITS reduced the amplitude of all conducting states (three subconductances and fully open). This effect was fully reversible upon SITS removal from the bath solution. A half-inhibitory concentration (IC₅₀) of 55.6 ± 2.7 μm (+60 mV) and 66.7 ± 2.2 (−60 mV) was obtained from the fitting to a Langmuir function. All these results are compatible with the existence of two SITS binding sites in the chloride channel: one of high affinity responsible for the increment in the number of transitions to subconductance states, and one low affinity binding site involved in the reduction of the amplitude of all conducting states. Received: 29 October 1998/Revised: 16 February 1999     

Identification of Multiple Gypsy LTR-Retrotransposon Lineages in Vertebrate Genomes

Gypsy LTR-retrotransposons have been identified in the genomes of many organisms, but only a small number of vertebrate examples have been reported to date. Here we show that members of this family are likely to be widespread in many vertebrate classes with the possible exceptions of mammals and birds. Phylogenetic analyses demonstrate that although there are several distinct lineages of vertebrate gypsy LTR-retrotransposons, the majority clusters into one monophyletic clade. Groups of fungal, plant, and insect elements were also observed, suggesting horizontal transfer between phyla may be infrequent. However, in contrast to this, there was little evidence to support sister relationships between elements derived from vertebrate and insect hosts. In fact, the majority of the vertebrate elements appeared to be most closely related to a group of gypsy LTR-retrotransposons present within fungi. This implies either that at least one horizontal transmission between these two phyla has occurred previously or that a gypsy LTR-retrotransposon lineage has been lost from insect taxa. Received: 22 December 1998 / Accepted: 6 April 1999     

Using Homolog Groups to Create a Whole-Genomic Tree of Free-Living Organisms: An Update

Genomic trees have been constructed based on the presence and absence of families of protein-encoding genes observed in 27 complete genomes, including genomes of 15 free-living organisms. This method does not rely on the identification of suspected orthologs in each genome, nor the specific alignment used to compare gene sequences because the protein-encoding gene families are formed by grouping any protein with a pairwise similarity score greater than a preset value. Because of this all inclusive grouping, this method is resilient to some effects of lateral gene transfer because transfers of genes are masked when the recipient genome already has a homolog (not necessarily an ortholog) of the incoming gene. Of 71 genes suspected to have been laterally transferred to the genome of Aeropyrum pernix, only approximately 7 to 15 represent genes where a lateral gene transfer appears to have generated homoplasy in our character dataset. The genomic tree of the 15 free-living taxa includes six different bacterial orders, six different archaeal orders, and two different eukaryotic kingdoms. The results are remarkably similar to results obtained by analysis of rRNA. Inclusion of the other 12 genomes resulted in a tree only broadly similar to that suggested by rRNA with at least some of the differences due to artifacts caused by the small genome size of many of these species. Very small genomes, such as those of the two Mycoplasma genomes included, fall to the base of the Bacterial domain, a result expected due to the substantial gene loss inherent to these lineages. Finally, artificial ``partial genomes' were generated by randomly selecting ORFs from the complete genomes in order to test our ability to recover the tree generated by the whole genome sequences when only partial data are available. The results indicated that partial genomic data, when sampled randomly, could robustly recover the tree generated by the whole genome sequences. Received: 30 May 2001 / Accepted: 10 October 2001     

On Negative Selection Against ATG Triplets Near Start Codons in Eukaryotic and Prokaryotic Genomes

The frequencies of ATG triplets in the genomes of various species were systematically analyzed, and the frequency of ATG triplets was significantly low around start codons in both prokaryotic and eukaryotic genomes. In eukaryotes, however, the frequency decrease before the start codon is much more evident than that after the start codon. In prokaryotes, on the other hand, the ATG frequency pattern around the start codon is less evident, and—more importantly—symmetric. We also computed average distances between a start codon and its nearest upstream-located ATG triplet and found a general tendency for the average distances to be longer in higher organisms. Received: 2 April 1998 / Accepted: 12 August 1998