Testing claims of gene conversion between multigene family members: examples from echinoderm actin genes

Molecular genetics studies often infer the occurrence of gene conversion events based on simple sequence similarity observations that do not include any statistical analyses. I show that the statistical significance of two previously proposed gene conversion events can easily be tested and point out that a variety of methods are available to perform gene conversion analyses. Received: 6 June 2001 / Accepted: 22 June 2001     

Evolutionary dynamics of the human endogenous retrovirus family HERV-K inferred from full-length proviral genomes

Several distinct families of endogenous retroviruses exist in the genomes of primates. Most of them are remnants of ancient germ-line infections. The human endogenous retrovirus family HERV-K represents the unique known case of endogenous retrovirus that amplified in the human genome after the divergence of human and chimpanzee lineages. There are two types of HERV-K proviral genomes differing by the presence or absence of 292 bp in the pol-env boundary. Human-specific insertions exist for both types. The analyses shown in the present work reveal that several lineages of type 1 and type 2 HERV-K proviruses remained transpositionally active after the human/chimpanzee split. The data also reflect the important role of mosaic evolution (either by recombination or gene conversion) during the evolutionary history of HERV-K. Received: 5 February 2001 / Accepted: 22 March 2001     

A Test of Amino Acid Reversibility

In studies of molecular evolution, the assumption that protein evolution is reversible has often been made, but rarely tested. Here we use a large set of orthologous murid protein coding sequences to perform a simple test of reversibility, and find no evidence to reject the assumption of reversibility in protein evolution. Received: 10 October 2000 / Accepted: 18 January 2001     

Phylogenetic Analysis of Invertebrate Lysozymes and the Evolution of Lysozyme Function

We isolated and sequenced the cDNAs coding for lysozymes of six bivalve species. Alignment and phylogenetic analysis showed that, together with recently described bivalve lysozymes, the leech destabilase, and a number of putative proteins from extensive genomic and cDNA analyses, they belong to the invertebrate type of lysozymes (i type), first described by Jollès and Jollès (1975). We determined the genomic structure of the gene encoding the lysozyme of Mytilus edulis, the common mussel. We provide evidence that the central exon of this gene is homologous to the second exon of the chicken lysozyme gene, belonging to the c type. We propose that the origin of this domain can be traced back in evolution to the origin of bilaterian animals. Phylogenetic analysis suggests that i-type proteins form a monophyletic family. Received: 21 May 2001 / Accepted: 22 October 2001     

Sucrose Phosphate Synthase Genes in Plants Belong to Three Different Families

We present phylogenetic analyses to demonstrate that there are three families of sucrose phosphate synthase (SPS) genes present in higher plants. Two data sets were examined, one consisting of full-length proteins and a second larger set that covered a highly conserved region including the 14-3-3 binding region and the UDPGlu active site. Analysis of both datasets showed a well supported separation of known genes into three families, designated A, B, and C. The genomic sequences of Arabidopsis thaliana include a member in each family: two genes on chromosome 5 belong to Family A, one gene on chromosome 1 to Family B, and one gene on chromosome 4 to Family C. Each of three Citrus genes belong to one of the three families. Intron/exon organization of the four Arabidopsis genes differed according to phylogenetic analysis, with members of the same family from different species having similar genomic organization of their SPS genes. The two Family A genes on Arabidopsis chromosome 5 appear to be due to a recent duplication. Analysis of published literature and ESTs indicated that functional differentiation of the families was not obvious, although B family members appear not to be expressed in roots. B family genes were cloned from two Actinidia species and southern analysis indicated the presence of a single gene family, which contrasts to the multiple members of Family A in Actinidia. Only two family C genes have been reported to date. Received: 17 April 2001 / Accepted: 27 August 2001     

Phylogenetic analysis of viroid and viroid-like satellite RNAs from plants: a reassessment

The proposed monophyletic origin of a group of subviral plant pathogens (viroids and viroid-like satellite RNAs), as well as the phylogenetic relationships and the resulting taxonomy of these entities, has been recently questioned. The criticism comes from the (apparent) lack of sequence similarity among these RNAs necessary to reliably infer a phylogeny. Here we show that, despite their low overall sequence similarity, a sequence alignment manually adjusted to take into account all the local similarities and the insertions/deletions and duplications/rearrangements described in the literature for viroids and viroid-like satellite RNA, along with the use of an appropriate estimator of genetic distances, constitutes a data set suitable for a phylogenetic reconstruction. When the likelihood-mapping method was applied to this data set, the tree-likeness obtained was higher than that corresponding to a sequence alignment that does not take into consideration the local similarities. In addition, bootstrap analysis also supports the major groups previously proposed and the reconstruction is consistent with the biological properties of this RNAs. Received: 17 January 2001 / Accepted: 16 March 2001     

Hepatitis C Virus Evolutionary Patterns Studied Through Analysis of Full-Genome Sequences

The evolutionary patterns of hepatitis C virus (HCV), including the best-fitting nucleotide substitution model and the molecular clock hypothesis, were investigated by analyzing full-genome sequences available in the HCV database. The likelihood ratio test allowed us to discriminate among different evolutionary hypotheses. The phylogeny of the six major HCV types was accurately inferred, and the final tree was rooted by reconstructing the hypothetical HCV common ancestor with the maximum likelihood method. The presence of phylogenetic noise and the relative nucleotide substitution rates in the different HCV genes were also examined. These results offer a general guideline for the future of HCV phylogenetic analysis and also provide important insights on HCV origin and evolution. Received: 13 January 2001 / Accepted: 21 June 2001     

Uneven Distribution of Repetitive Trinucleotide Motifs in Human Immunoglobulin Heavy Variable Genes

Insertions and deletions of entire codons have recently been discovered as a mechanism by which B cells, in addition to conventional base substitution, evolve the antibodies produced by their immunoglobulin genes. These events frequently seem to involve repetitive sequence motifs in the antibody-encoding genes, and it has been suggested that they occur through polymerase slippage. In order to better understand the process of codon deletion, we have analyzed the human immunoglobulin heavy variable (IGHV) germline gene repertoire for the presence of trinucleotide repeats. Such repeats would ensure that the reading frame is maintained in the case of a deletional event, as slippage over multiples of three bases would be favored. We demonstrate here that IGHV genes specifically carry repetitive trinucleotide motifs in the complementarity-determining regions (CDR) 1 and 2, thus making these parts of the genes that encode highly flexible structures particularly prone to functional deletions. We propose that the human IGHV repertoire carries inherent motifs that allow an antibody response to develop efficiently by targeting codon deletion events to the parts of the molecule that are likely to be able to harbor such modifications. Received: 10 April 2001 / Accepted: 27 August 2001     

Maximum Likelihood Methods Reveal Conservation of Function Among Closely Related Kinesin Families

We have reconstructed the evolution of the anciently derived kinesin superfamily using various alignment and tree-building methods. In addition to classifying previously described kinesins from protists, fungi, and animals, we analyzed a variety of kinesin sequences from the plant kingdom including 12 from Zea mays and 29 from Arabidopsis thaliana. Also included in our data set were four sequences from the anciently diverged amitochondriate protist Giardia lamblia. The overall topology of the best tree we found is more likely than previously reported topologies and allows us to make the following new observations: (1) kinesins involved in chromosome movement including MCAK, chromokinesin, and CENP-E may be descended from a single ancestor; (2) kinesins that form complex oligomers are limited to a monophyletic group of families; (3) kinesins that crosslink antiparallel microtubules at the spindle midzone including BIMC, MKLP, and CENP-E are closely related; (4) Drosophila NOD and human KID group with other characterized chromokinesins; and (5) Saccharomyces SMY1 groups with kinesin-I sequences, forming a family of kinesins capable of class V myosin interactions. In addition, we found that one monophyletic clade composed exclusively of sequences with a C-terminal motor domain contains all known minus end-directed kinesins. Received: 20 February 2001 / Accepted: 5 June 2001     

Reversible Electropermeabilization of Mammalian Cells by High-Intensity, Ultra-Short Pulses of Submicrosecond Duration

Mouse myeloma cells were electropermeabilized by single square-wave electric pulses with amplitudes of up to ∼150 kV/cm and durations of 10–100 nsec. The effects of the field intensity, pulse duration and medium conductivity on cell viability and field-induced uptake of molecules were analyzed by quantitative flow cytometry using the membrane-impermeable fluorescent dye propidium iodide as indicator molecule. Despite the extremely large field strengths, the majority of cells survived the exposure to ultra-short field pulses. The electrically induced dye uptake increased markedly with decreasing conductivity of the suspending medium. We assigned this phenomenon to the transient electrodeformation (stretching) force that assumes its maximum value if cells are suspended in low-conductivity media, i.e., if the external conductivity σ_e is smaller than that of the cytosol σ_i. The stretching force vanishes when σ_e is equal to or larger than σ_i. Due to their capability of delivering extremely large electric fields, the pulse power systems used here appear to be a promising tool for the electropermeabilization of very small cells and vesicles (including intracellular organelles, liposomes, etc.). Received: 15 May 2001/Revised: 20 July 2001     

Simultaneous Molecular and Morphological Analysis of Braconid Relationships (Insecta: Hymenoptera: Braconidae) Indicates Independent mt-tRNA Gene Inversions Within a Single Wasp Family

We investigated the phylogeny of the Braconidae (Insecta: Hymenoptera) with a much expanded data set compared with that of previous attempts, employing 16S and 28S rDNA gene fragments, together with a suite of morphological characters, from 74 ingroup taxa. Most notably, parsimony analyses under a range of models recovered the Aphidiinae as sister group to the cyclostomes and the Ichneutinae as sister group to the microgastroids. The cyclostomes were recovered as a natural group only if certain, putatively misplaced genera (Mesostoa, Aspilodemon) were excluded from them. Further, mapping of rearrangement characters onto this phylogeny of the Braconidae indicated parallel inversions of the mt-tRNA^D gene, with the two instances of inversion distinguishable by the presence or absence of an additional tRNA gene (tRNA^H). This is the first report of a parallel inversion of a mt-tRNA gene and makes the Braconidae the first metazoan family to display both parallel inversions and translocations. Received: 6 April 2001 / Accepted: 9 July 2001     

The Evolutionary History of Prosaposin: Two Successive Tandem-Duplication Events Gave Rise to the Four Saposin Domains in Vertebrates

Prosaposin is a multifunctional protein encoded by a single-copy gene. It contains four saposin domains (A, B, C, and D) occurring as tandem repeats connected by linker sequences. Because the saposin domains are similar to one another, it is deduced that they were created by sequential duplications of an ancestral domain. There are two types of evolutionary scenarios that may explain the creation of the four-domain gene: (1) two rounds of tandem internal gene duplication and (2) three rounds of duplications. An evolutionary and phylogenetic analysis of saposin DNA and amino acid sequences from human, mouse, rat, chicken, and zebrafish indicates that the first evolutionary scenario is the most likely. Accordingly, an ancestral saposin-unit duplication produced a two-domain gene, which, subsequently, underwent a second complete tandem duplication to give rise to the present four-domain structure of the prosaposin gene. Received: 8 February 2001 / Accepted: 29 June 2001     

Phylogeny Derived from Coding Retroviral Genome Organization

When divergence between viral species is large, the analysis and comparison of nucleotide or protein sequences are dependent on mutation biases and multiple substitutions per site leading, among other things, to the underestimation of branch lengths in phylogenetic trees. To avoid the problem of multiply substituted sites, a method not directly based on the nucleic or protein sequences has been applied to retroviruses. It consisted of asking questions about genome structure or organization, and gene function, the series of answers creating coded sequences analyzed by phylogenic software. This method recovered the principal retroviral groups such as the lentiviruses and spumaviruses and highlighted questions and answers characteristic of each group of retroviruses. In general, there was reasonable concordance between the coded genome methodology and that based on conventional phylogeny of the integrase protein sequence, indicating that integrase was fixing mutations slowly enough to marginalize the problem of multiple substitutions at sites. To a first approximation, this suggests that the acquisition of novel genetic features generally parallels the fixation of amino acid substitutions. Received: 18 May 2001 / Accepted: 7 September 2001     

The Chemical Basis of Membrane Bioenergetics

All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents. Received: 6 June 2001 / Accepted: 16 October 2001     

Lineage Specificity of the Evolutionary Dynamics of the mtDNA D-Loop Region in Rodents

This paper reports an intraorder study on the D-loop-containing region of the mitochondrial DNA in rodents. A complete multialignment of this region is not feasible with the exception of some conserved regions. The comparative analysis of 25 complete rodent sequences from 23 species plus one lagomorph has revealed that only the central domain (CD), a conserved region of about 80 bp in the extended termination-associated sequences (ETAS) domain, adjacent to the CD, the ETAS1, and conserved sequence block (CSB) 1 blocks are present in all rodent species, whereas the presence of CSB2 and CSB3 is erratic within the order. We have also found a conserved region of 90 bp located between tRNAPro and ETAS1 present in fat dormouse, squirrel, guinea pig, and rabbit. Repeated sequences are present in both the ETAS and the CSB domain, but the repeats differ in length, copy number, and base composition in different species. The potential use of the D-loop for evolutionary studies has been investigated; the presence/absence of conserved blocks and/or repeated sequences cannot be used as a reliable phylogenetic marker, since in some cases they may be shared by distantly related organisms but not by close ones, while in other ones a relationship between tree topology and presence/absence of such motifs is observed. Better results can be obtained by the use of the CD, which, however, due to its reduced size, when used for tracing a phylogenetic tree, shows some nodes with low statistical support. Received: 26 February 2001 / Accepted: 6 June 2001     

Evolution of MADS-box gene induction by FLO/LFY genes

Some MADS-box genes function as floral homeotic genes. The Arabidopsis LFY gene is a positive regulator of floral homeotic genes, and homologs of the FLO/LFY gene family in other angiosperms and gymnosperms are likely to have a similar function. To investigate the origin of the floral homeotic gene regulatory cascade involving the FLO/LFY gene, FLO/LFY homologs were cloned from a leptosporangiate fern (Ceratopteris richardii), two eusporangiate ferns (Angiopteris lygodiifolia and Botrychium multifidum var. robustum), three fern allies (Psilotum nudum, Equisetum arvense, and Isoetes asiatica), and a moss (Physcomitrella patens). The FLO/LFY gene phylogenetic tree indicates that both duplication and loss of FLO/LFY homologs occurred during the course of vascular plant evolution. The expression patterns of the Ceratopteris LFY genes (CrLFY1 and 2) were assessed. CrLFY1 expression was prominent in tissues including shoot tips and circinate reproductive leaves, but very weak in other tissues examined. Expression of CrLFY2 was also prominent in tissues, including shoot tips and circinate reproductive leaves. These patterns of expression are dissimilar to that of any Ceratopteris MADS-box gene previously reported, suggesting that the induction of MADS-box genes by FLO/LFY is not established at the stage of ferns. Received: 4 January 2001 / Accepted: 28 February 2001     

Taking Variation of Evolutionary Rates Between Sites into Account in Inferring Phylogenies

As methods of molecular phylogeny have become more explicit and more biologically realistic following the pioneering work of Thomas Jukes, they have had to relax their initial assumption that rates of evolution were equal at all sites. Distance matrix and likelihood methods of inferring phylogenies make this assumption; parsimony, when valid, is less limited by it. Nucleotide sequences, including RNA sequences, can show substantial rate variation; protein sequences show rates that vary much more widely. Assuming a prior distribution of rates such as a gamma distribution or lognormal distribution has deservedly been popular, but for likelihood methods it leads to computational difficulties. These can be resolved using hidden Markov model (HMM) methods which approximate the distribution by one with a modest number of discrete rates. Generalized Laguerre quadrature can be used to improve the selection of rates and their probabilities so as to more nearly approach the desired gamma distribution. A model based on population genetics is presented predicting how the rates of evolution might vary from locus to locus. Challenges for the future include allowing rates at a given site to vary along the tree, as in the ``covarion' model, and allowing them to have correlations that reflect three-dimensional structure, rather than position in the coding sequence. Markov chain Monte Carlo likelihood methods may be the only practical way to carry out computations for these models. Received: 8 February 2001 / Accepted: 20 May 2001     

Identifying a Folding Nucleus for the Lysozyme/α-Lactalbumin Family from Sequence Conservation Clusters

Four subfamilies of c-type lysozyme and one subfamily of α-lactalbumin are defined from 78 sequences, and their folding nucleus is identified with a method based on conserved residues and native structural contacts between pairs of conserved residues. One large cluster of 19 conserved residues is found which is mostly nonpolar, buried, and nonfunctional. It can be subdivided into three subclusters: (1) conserved residues in four helices; (2) conserved residues that stabilize the connector between the α and the β domains; and (3) a β-turn, sitting in the middle of a bowl of α-helix residues. It is proposed that this folding nucleus initiates four helices, A, B, C, and D, three β sheets, and the connector, which corresponds closely to the nucleation of the so-called fast folding track pathway. As the secondary structures propagate, nonconserved residues and functionally conserved residues would form additional contacts. The conserved residues are selected with a phylogenetic scheme in which single members of subfamilies are selected. Subfamilies are then equally weighted to obtain the consensus conservation. Received: 11 June 2001 / Accepted: 28 August 2001     

The Differential Killing of Genes by Inversions in Prokaryotic Genomes

We have elaborated a method which has allowed us to estimate the direction of translocation of orthologs which have changed, during the phylogeny, their positions on chromosome in respect to the leading or lagging role of DNA strands. We have shown that the relative number of translocations which have switched positions of genes from the leading to the lagging DNA strand is lower than the number of translocations which have transferred genes from the lagging strand to the leading strand of prokaryotic genomes. This paradox could be explained by assuming that the stronger mutation pressure and selection after inversion preferentially eliminate genes transferred from the leading to the lagging DNA strand. Received: 12 December 2000 / Accepted: 20 April 2001     

Selective Pressures on RNA Hairpins In Vivo and In Vitro

Comparison of the most stable potential hairpins in the sequences of natural ribozymes with those in the randomized sequences has revealed that the hairpin loop energies are lower than expected by chance. Although these hairpins are not necessarily parts of functional structures, there is a selective pressure to diminish the destabilizing free energies of the hairpin loops. In contrast, no significant bias is observed in the stacking values of the most stable stems. In the ribozymes isolated in vitro the loops of potential hairpins are closer to random values, which can result in less efficient folding rates. Furthermore, the effects of kinetic traps seem to be more significant in the folding pathways of the in vitro isolates due to a potential to form stable stacks incompatible with the functional folds. Similarly to natural ribozyme sequences, the untranslated regions of viral RNAs also form hairpins with relatively low loop free energies. These evolutionary trends suggest ways for efficient engineering of improved RNA constructs on the basis of analysis of in vitro isolates and approaches for the search of regions coding for functional RNA structures in large genome sequences. Received: 12 January 2001 / Accepted: 21 May 2001