A branch-heterogeneous model of protein evolution for efficient inference of ancestral sequences.

Most models of nucleotide or amino acid substitution used in phylogenetic studies assume that the evolutionary process has been homogeneous across lineages and that composition of nucleotides or amino acids has remained the same throughout the tree. These oversimplified assumptions are refuted by the observation that compositional variability characterizes extant biological sequences. Branch-heterogeneous models of protein evolution that account for compositional variability have been developed, but are not yet in common use because of the large number of parameters required, leading to high computational costs and potential overparameterization. Here, we present a new branch-nonhomogeneous and nonstationary model of protein evolution that captures more accurately the high complexity of sequence evolution. This model, henceforth called Correspondence and likelihood analysis (COaLA), makes use of a correspondence analysis to reduce the number of parameters to be optimized through maximum likelihood, focusing on most of the compositional variation observed in the data. The model was thoroughly tested on both simulated and biological data sets to show its high performance in terms of data fitting and CPU time. COaLA efficiently estimates ancestral amino acid frequencies and sequences, making it relevant for studies aiming at reconstructing and resurrecting ancestral amino acid sequences. Finally, we applied COaLA on a concatenate of universal amino acid sequences to confirm previous results obtained with a nonhomogeneous Bayesian model regarding the early pattern of adaptation to optimal growth temperature, supporting the mesophilic nature of the Last Universal Common Ancestor.

Genetic mapping of a major dominant gene for resistance to Ralstonia solanacearum in eggplant.

Resistance of eggplant against Ralstonia solanacearum phylotype I strains was assessed in a F(6) population of recombinant inbred lines (RILs) derived from a intra-specific cross between S. melongena MM738 (susceptible) and AG91-25 (resistant). Resistance traits were determined as disease score, percentage of wilted plants, and stem-based bacterial colonization index, as assessed in greenhouse experiments conducted in Réunion Island, France. The AG91-25 resistance was highly efficient toward strains CMR134, PSS366 and GMI1000, but only partial toward the highly virulent strain PSS4. The partial resistance found against PSS4 was overcome under high inoculation pressure, with heritability estimates from 0.28 to 0.53, depending on the traits and season. A genetic map was built with 119 AFLP, SSR and SRAP markers positioned on 18 linkage groups (LG), for a total length of 884 cM, and used for quantitative trait loci (QTL) analysis. A major dominant gene, named ERs1, controlled the resistance to strains CMR134, PSS366, and GMI1000. Against strain PSS4, this gene was not detected, but a significant QTL involved in delay of disease progress was detected on another LG. The possible use of the major resistance gene ERs1 in marker-assisted selection and the prospects offered for academic studies of a possible gene for gene system controlling resistance to bacterial wilt in solanaceous plants are discussed.

Experimental assessment of the accuracy of genomic selection in sugarcane.

Sugarcane cultivars are interspecific hybrids with an aneuploid, highly heterozygous polyploid genome. The complexity of the sugarcane genome is the main obstacle to the use of marker-assisted selection in sugarcane breeding. Given the promising results of recent studies of plant genomic selection, we explored the feasibility of genomic selection in this complex polyploid crop. Genetic values were predicted in two independent panels, each composed of 167 accessions representing sugarcane genetic diversity worldwide. Accessions were genotyped with 1,499 DArT markers. One panel was phenotyped in Reunion Island and the other in Guadeloupe. Ten traits concerning sugar and bagasse contents, digestibility and composition of the bagasse, plant morphology, and disease resistance were used. We used four statistical predictive models: bayesian LASSO, ridge regression, reproducing kernel Hilbert space, and partial least square regression. The accuracy of the predictions was assessed through the correlation between observed and predicted genetic values by cross validation within each panel and between the two panels. We observed equivalent accuracy among the four predictive models for a given trait, and marked differences were observed among traits. Depending on the trait concerned, within-panel cross validation yielded median correlations ranging from 0.29 to 0.62 in the Reunion Island panel and from 0.11 to 0.5 in the Guadeloupe panel. Cross validation between panels yielded correlations ranging from 0.13 for smut resistance to 0.55 for brix. This level of correlations is promising for future implementations. Our results provide the first validation of genomic selection in sugarcane.

Species identification of staphylococci by amplification and sequencing of the tuf gene compared to the gap gene and by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Staphylococcal species, notably, coagulase-negative staphylococci (CoNS), are frequently misidentified using phenotypic methods. The partial nucleotide sequences of the tuf and gap genes were determined in 47 reference strains to assess their suitability, practicability, and discriminatory power as target molecules for staphylococcal identification. The partial tuf gene sequence was selected and further assessed with a collection of 186 strains, including 35 species and subspecies. Then, to evaluate the efficacy of this genotyping method versus the technology of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS), the 186 strains were identified using MALDI-TOF-MS (Axima® Shimadzu) coupled to the SARAMIS® database (AnagnosTec). The French National Reference Center for Staphylococci identification method was used as a reference. One hundred and eighty-four strains (98.9%) were correctly identified by tuf gene sequencing. Only one strain was misidentified and one was unidentified. MALDI-TOF-MS identified correctly 138 isolates (74.2%). Four strains were misidentified, 39 were unidentified, five were identified at the group (hominis/warneri) level, and one strain was identified at the genus level. These results confirm the value of MALDI-TOF-MS identification for common species in clinical laboratory practice and the value of the partial tuf gene sequence for the identification of all staphylococcal species as required in a reference laboratory.

A nonhyperthermophilic common ancestor to extant life forms.

The G+C nucleotide content of ribosomal RNA (rRNA) sequences is strongly correlated with the optimal growth temperature of prokaryotes. This property allows inference of the environmental temperature of the common ancestor to all life forms from knowledge of the G+C content of its rRNA sequences. A model of sequence evolution, assuming varying G+C content among lineages and unequal substitution rates among sites, was devised to estimate ancestral base compositions. This method was applied to rRNA sequences of various species representing the major lineages of life. The inferred G+C content of the common ancestor to extant life forms appears incompatible with survival at high temperature. This finding challenges a widely accepted hypothesis about the origin of life.

Nrh, a human homologue of Nr-13 associates with Bcl-Xs and is an inhibitor of apoptosis.

In search of human homologues of the anti-apoptotic protein Nr-13, we have characterized a human EST clone that potentially encodes a protein, which is the closest homologue of Nr-13 among the Bcl-2 family members, to date known, in humans. Phylogenetic analyses suggest Human nrh, Mouse diva/boo and Quail nr-13 to be orthologous genes. The nrh gene has the same overall organization as nr-13 and diva/boo with one single intron interrupting the ORF at the level of the Bcl-2-homology domain BH2. RT-PCR-based analysis of nrh expression indicated that this gene is preferentially expressed in the lungs, the liver and the kidneys. Interestingly, two in frame ATG codons can lead potentially to the synthesis of two products, one of them lacking 10 aminoacids at the N-terminal end. Sequence alignment with Nr-13 and Diva/Boo in addition to secondary structure prediction of the nrh transcript suggested that the shortest protein will be preferentially synthetized. Immunohistochemical analyses have revealed that Nrh is associated with mitochondria and the nuclear envelope. Moreover, Nrh preferentially associates with the apoptosis accelerator Bcl-Xs and behaves as an inhibitor of apoptosis both in yeast and vertebrate cells.

Evolution of the primate beta-globin gene region. High rate of variation in CpG dinucleotides and in short repeated sequences between man and chimpanzee.

A 5500 base-pair fragment including the beta-globin gene downstream from codon 122 and about 4000 base-pairs of its 5' flanking sequence was cloned from chimpanzee DNA and thoroughly sequenced before being compared with the corresponding human sequence: 88 point differences (83 substitutions and 5 deletions or insertions of 1 base-pair) were detected as well as seven more important deletion/insertion events. These changes occur preferentially in two kinds of structure. First, 40% of the CpG dinucleotides present in either human or chimpanzee sequences are affected by nucleotide variations. This corresponds to a divergence level considerably higher than that expected. Second, most short repeated sequences found in the 5' extragenic sequence are involved in mutational events (amplification or contraction of the number of basic motifs as well as point substitutions or deletions/insertions of 1 base-pair). Considering the very low level of nucleotide sequence divergence between these two closely related species, our data provide direct evidence for CpG and tandem array instability.

Synthetic approaches to a mononucleotide prodrug of cytarabine.

Synthetic pathways to a mononucleotide prodrug of cytarabine (Ara-C) bearing S-pivaloyl-2-thioethyl (tBuSATE) groups, as biolabile phosphate protections, are reported. Using a common phosphoramidite approach, two different kinds of nucleoside protecting groups have been investigated. During this study, we observed an intermolecular migration of the Boc protecting group in the course of the tert-butyldimethylsilyl ether cleavage using tetrabutyl ammonium fluoride.

Isolation and characterization of a cDNA encoding a chicken actin-like protein.

We report the isolation and characterization of a chicken cDNA which putatively encodes an actin-like protein (chACTL). This 394-amino-acid (aa) polypeptide shares sequence homology (81, 70 and 67% identical aa, respectively) with three actin-related proteins (ARP) described for Drosophila melanogaster (ARP14D), Caenorhabditis elegans (ACTL) and Saccharomyces cerevisiae (ACT2). At least six chACTL transcripts were detected in different tissues during chick embryogenesis. Sequence analysis suggests that at least three groups of ARP have been evolutionarily conserved.

WWW-query: an on-line retrieval system for biological sequence banks.

We have developed a World Wide Web (WWW) version of the sequence retrieval system Query: WWW-Query. This server allows to query nucleotide sequence banks in the EMBL/GenBank/DDBJ formats and protein sequence banks in the NBRF/PIR format. WWW-Query includes all the features of the on-line sequences browsers already available: possibility to build complex queries, integration of cross-references with different data banks, and access to the functional zones of biological interest. It also provides original services not available elsewhere: introduction of the notion of re-usable sequence lists, integration of dedicated helper applications for visualizing alignments and phylogenetic trees and links with multivariate methods for studying codon usage or for complementing phylogenies.

Non-parametric statistics for nucleic acid sequence study.

The use of non-parametric statistics for nucleic acid sequence studies is illustrated by some examples. This method is highly flexible and allows design of specific tests for detecting sequence structure. Tests devoted to local repetitivity, codon nearest neighbors, and dinucleotide avoidance are discussed in detail. An appendix indicates all computations required to use these tests.

System analysis and nucleic acid sequence banks.

The mass of published nucleic acid sequence data has required the design of several computerized data bases. We show that this activity is related to the methodology of System Analysis and that data bases are a means of modeling biological knowledge. As an example, the ACNUC data base we have created is presented.

[Prediction of secondary structures of nucleic acids: algorithmic and physical aspects]. / Prédiction des structures secondaires dans les acides nucléiques: aspects algorithmiques et physiques.

Prediction of secondary structures in nucleic acids requires both an adequate physical model and powerful calculation algorithms. In our approach, we cut the molecules in sections of which the contributions to the global energy are context-dependent but roughly additive. The structure of minimum energy is obtained by a tree search under constraints of binary incompatibilities. Our algorithm of the "incompatibility islets" is shown to be more powerful than the "bit parallel forward checking" algorithm, well known in Artificial Intelligence. Recurrent algorithms, proposed by other authors are even more rapid, but often miss the correct structures, for they demand a strict additivity of the energetic contributions, physically unjustified. New strategies, required to deal with molecules of more than 200 nucleotides are discussed. Our physical model has been improved by considering the special case of internal loops beginning with a G-A opposition. A bonus of 1.5 kcal. is attributed to such a feature, at each side of an internal loop. To illustrate our programs, we give the computed schemes for the 3' termini of the small subunit ribosomal RNA.

Molecular phylogeny of Eubacteria: a new multiple tree analysis method applied to 15 sequence data sets questions the monophyly of gram-positive bacteria.

Phylogenetic relationships between the major eubacterial phyla were studied using the sequences of 15 homologous bacterial genes. Neither the classical concatenation strategy nor a new multiple tree analysis method involving statistical tests of the inferred phylogenetic relationships provided any (solid) conclusions about eubacterial phylogeny; no pairs of eubacterial phyla proved to be closer to each other in the 15 reconstructed trees than would be expected for trees with random topologies. The phylogeny of Eubacteria therefore appears to be tightly bush-like. Moreover, results from both concatenation and multiple tree analysis raise doubts concerning the monophyly of the so-called Gram-positive bacteria phylum, since the monophyly hypothesis is no more strongly supported by data than its alternatives. It is noteworthy that the structural bases for the Gram-positive phenotype are not incompatible with the hypothesis of independent emergence of this character at two different times.

Bacterial molecular phylogeny using supertree approach.

It has been claimed that complete genome sequences would clarify phylogenetic relationships between organisms but, up to now, no satisfying approach has been proposed to use efficiently these data. For instance, if the coding of presence or absence of genes in complete genomes gives interesting results, it does not take into account the phylogenetic information contained in sequences and ignores hidden paralogy by using a similarity-based definition of orthology. Also, concatenation of sequences of different genes takes hardly in consideration the specific evolutionary rate of each gene. At last, building a consensus tree is strongly limited by the low number of genes shared among all organisms. Here, we use a new method based on supertree construction, which permits to cumulate in one supertree the information and statistical support of hundreds of trees from orthologous gene families and to build the phylogeny of 33 prokaryotes and four eukaryotes with completely sequenced genomes. This approach gives a robust supertree, which demonstrates that a phylogeny of prokaryotic species is conceivable and challenges the hypothesis of a thermophilic origin of bacteria and present-day life. The results are compatible with the hypothesis of a core of genes for which lateral transfers are rare but they raise doubts on the widely admitted "complexity hypothesis" which predicts that this core is mainly implicated in informational processes.

Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation.

With today's technology for production of molecular sequences, DNA taxonomy and barcoding arose as a new tool for evolutionary biology and ecology. However, their validities still need to be empirically evaluated. Of most importance is the strength of the correlation between morphological taxonomy and molecular divergence and the possibility to define some molecular thresholds. Here, we report measurements of this correlation for two mitochondrial genes (COI and 16S rRNA) within the sub-phylum Crustacea. Perl scripts were developed to ensure objectivity, reproducibility, and exhaustiveness of our tests. Our analysis reveals a general correlation between molecular divergence and taxonomy. This correlation is particularly high for shallow taxonomic levels allowing us to propose a COI universal crustacean threshold to help species delimitation. At higher taxonomic levels this correlation decreases, particularly when comparing different families. Those results plead for DNA use in taxonomy and suggest an operational method to help crustacean species delimitation that is linked to the phylogenetic species definition. This pragmatic tool is expected to fine tune the present classification, and not, as some would have believed, to tear it apart.

Phylogeography of a subterranean amphipod reveals cryptic diversity and dynamic evolution in extreme environments.

Extreme conditions in subsurface are suspected to be responsible for morphological convergences, and so to bias biodiversity assessment. Subterranean organisms are also considered as having poor dispersal abilities that in turn generate a large number of endemic species when habitat is fragmented. Here we test these general hypotheses using the subterranean amphipod Niphargus virei. All our phylogenetic analyses (Bayesian, maximum likelihood and distance), based on two independent genes (28S and COI), revealed the same tripartite structure. N. virei populations from Benelux, Jura region and the rest of France appeared as independent evolutionary units. Molecular rates estimated via global or Bayesian relaxed clock suggest that this split is at least 13 million years old and accredit the cryptic diversity hypothesis. Moreover, the geographical distribution of these lineages showed some evidence of recent dispersal through apparent vicariant barrier. In consequence, we argue that future analyses of evolution and biogeography in subsurface, or more generally in extreme environments, should consider dispersal ability as an evolving trait and morphology as a potentially biased marker.