Genomic resources and their influence on the detection of the signal of positive selection in genome scans.

Genome scans represent powerful approaches to investigate the action of natural selection on the genetic variation of natural populations and to better understand local adaptation. This is very useful, for example, in the field of conservation biology and evolutionary biology. Thanks to Next Generation Sequencing, genomic resources are growing exponentially, improving genome scan analyses in non-model species. Thousands of SNPs called using Reduced Representation Sequencing are increasingly used in genome scans. Besides, genome sequences are also becoming increasingly available, allowing better processing of short-read data, offering physical localization of variants, and improving haplotype reconstruction and data imputation. Ultimately, genome sequences are also becoming the raw material for selection inferences. Here, we discuss how the increasing availability of such genomic resources, notably genome sequences, influences the detection of signals of selection. Mainly, increasing data density and having the information of physical linkage data expand genome scans by (i) improving the overall quality of the data, (ii) helping the reconstruction of demographic history for the population studied to decrease false-positive rates and (iii) improving the statistical power of methods to detect the signal of selection. Of particular importance, the availability of a high-quality reference genome can improve the detection of the signal of selection by (i) allowing matching the potential candidate loci to linked coding regions under selection, (ii) rapidly moving the investigation to the gene and function and (iii) ensuring that the highly variable regions of the genomes that include functional genes are also investigated. For all those reasons, using reference genomes in genome scan analyses is highly recommended.

Evidence for a genetic sex determination in Cnidaria, the Mediterranean red coral (Corallium rubrum).

Sexual reproduction is widespread among eukaryotes, and the sex-determining processes vary greatly among species. While genetic sex determination (GSD) has been intensively described in bilaterian species, no example has yet been recorded among non-bilaterians. However, the quasi-ubiquitous repartition of GSD among multicellular species suggests that similar evolutionary forces can promote this system, and that these forces could occur also in non-bilaterians. Studying sex determination across the range of Metazoan diversity is indeed important to understand better the evolution of this mechanism and its lability. We tested the existence of sex-linked genes in the gonochoric red coral (Corallium rubrum, Cnidaria) using restriction site-associated DNA sequencing. We analysed 27 461 single nucleotide polymorphisms (SNPs) in 354 individuals from 12 populations including 53 that were morphologically sexed. We found a strong association between the allele frequencies of 472 SNPs and the sex of individuals, suggesting an XX/XY sex-determination system. This result was confirmed by the identification of 435 male-specific loci. An independent test confirmed that the amplification of these loci enabled us to identify males with absolute certainty. This is the first demonstration of a GSD system among non-bilaterian species and a new example of its convergence in multicellular eukaryotes.

let-756, a C. elegans fgf essential for worm development.

In vertebrates, Fibroblast Growth Factors (FGFs) and their receptors are involved in various developmental and pathological processes, including neoplasia. The number of FGFs and their large range of activities have made the understanding of their precise functions difficult. Investigating their biology in other species might be enlightening. A sequence encoding a putative protein presenting 30-40% identity with the conserved core of vertebrate FGFs has been identified by the C. elegans sequencing consortium. We show here that this gene is transcribed and encodes a putative protein of 425 amino acids (aa). The gene is expressed at all stages of development beyond late embryogenesis, peaking at the larval stages. Loss-of-function mutants of the let-756 gene are rescued by the wild type fgf gene in germline transformation experiments. Two partial loss-of-function alleles, s2613 and s2809, have a mutation that replaces aa 317 by a stop. The truncated protein retains the FGF core but lacks a C-termins portion. These worms are small and develop slowly into clear and scrawny, yet viable and fertile adults. A third allele, s2887, is inactivated by an inversion that disrupts the first exon. It causes a developmental arrest early in the larval stages. Thus, in contrast to the other nematode fgf gene egl-17, let-756/fgf is essential for worm development.

A rigorous method for multigenic families' functional annotation: the peptidyl arginine deiminase (PADs) proteins family example.

BACKGROUND: large scale and reliable proteins' functional annotation is a major challenge in modern biology. Phylogenetic analyses have been shown to be important for such tasks. However, up to now, phylogenetic annotation did not take into account expression data (i.e. ESTs, Microarrays, SAGE, ...). Therefore, integrating such data, like ESTs in phylogenetic annotation could be a major advance in post genomic analyses. We developed an approach enabling the combination of expression data and phylogenetic analysis. To illustrate our method, we used an example protein family, the peptidyl arginine deiminases (PADs), probably implied in Rheumatoid Arthritis. RESULTS: the analysis was performed as follows: we built a phylogeny of PAD proteins from the NCBI's NR protein database. We completed the phylogenetic reconstruction of PADs using an enlarged sequence database containing translations of ESTs contigs. We then extracted all corresponding expression data contained in EST database This analysis allowed us 1/To extend the spectrum of homologs-containing species and to improve the reconstruction of genes' evolutionary history. 2/To deduce an accurate gene expression pattern for each member of this protein family. 3/To show a correlation between paralogous sequences' evolution rate and pattern of tissular expression. CONCLUSION: coupling phylogenetic reconstruction and expression data is a promising way of analysis that could be applied to all multigenic families to investigate the relationship between molecular and transcriptional evolution and to improve functional annotation.

Monoclonal antibodies to antitumor Vinca alkaloids: thermodynamics and kinetics.

Spleen cells from a mouse and a rat immunized with vinblastine coupled to bovine serum albumin were fused in two independent experiments with P3 X 63-Ag8 (non-secreting variant) mouse myeloma cells. Three mouse X mouse (Vinca 1-3) and two rat X mouse (Vinca 4 and 5) hybrids were selected for production of Vinca alkaloid binding monoclonal antibodies. Each antibody had characteristic cross-reactivities with alkaloids structurally related to vinblastine: Vinca 1 reacted preferentially with deacetylated alkaloids (deacetyl vinblastine and vindesine) and Vinca 2 had a higher affinity for vinblastine and vincristine. Vinca 3-5 recognized equally vinblastine, vincristine and vindesine but differed with respect to their affinities for other analogues. No significant cross-reactivity of the monomeric alkaloids vindoline or catharanthine was observed with any antibody, and dimeric alkaloids modified in the catharanthine moiety had reduced immunoreactivity. Mouse monoclonal antibodies (Vinca 1 and 3) showed moderate affinity (2.2 X 10(-7) and 5.8 X 10(-9) M) for their respective best ligands and fast kinetics (dissociation rate constants greater than 3 X 10(-3) sec-1). Vinca 4 and 5, derived from the rat X mouse hybrids, had much higher affinities (1.5 X 10(-11) and 1.1 X 10(-11) M) and slower kinetics (dissociation rate constants: 2.4 X 10(-5) and 7.2 X 10(-6) sec-1). The major difference between these two antibodies was that Vinca 4 binds and releases the antigen more rapidly than Vinca 5 does. Somatic hybridization techniques thus generated monoclonal antibodies recognizing a given class of low mol. wt antigens with variable specificity, affinity and kinetic behavior, allowing the selection of reagents most appropriate for particular immunochemical applications.

The red coral (Corallium rubrum) transcriptome: a new resource for population genetics and local adaptation studies.

The question of species survival and evolution in heterogeneous environments has long been a subject for study. Indeed, it is often difficult to identify the molecular basis of adaptation to contrasted environments, and nongenetic effects increase the difficulty to disentangle fixed effects, such as genetic adaptation, from variable effects, such as individual phenotypic plasticity, in adaptation. Nevertheless, this question is also of great importance for understanding the evolution of species in a context of climate change. The red coral (Corallium rubrum) lives in the Mediterranean Sea, where at depths ranging from 5 to 600 m, it meets very contrasted thermal conditions. The shallowest populations of this species suffered from mortality events linked with thermal anomalies that have highlighted thermotolerance differences between individuals. We provide here a new transcriptomic resource, as well as candidate markers for the study of local adaptation. We sequenced the transcriptome of six individuals from 5 m and six individuals from 40 m depth at the same site of the Marseilles bay, after a period of common garden acclimatization. We found differential expression maintained between the two depths even after common garden acclimatization, and we analysed the polymorphism pattern of these samples. We highlighted contigs potentially implicated in the response to thermal stress, which could be good candidates for the study of thermal adaptation for the red coral. Some of these genes are also involved in the response to thermal stress in other corals. Our method enables the identification of candidate loci of local adaptation useful for other nonmodel organisms.

Myelin oligodendrocyte glycoprotein (MOG) gene polymorphisms and multiple sclerosis: no evidence of disease association with MOG.

The region surrounding the myelin oligodendrocyte glycoprotein (MOG) gene, located telomeric to the major histocompatibility complex on chromosome 6, was shown to contain three highly informative microsatellites. To examine the potential role of variants of the MOG gene in susceptibility to multiple sclerosis, these CA-repeat polymorphic markers were characterized on a sample of 169 multiple sclerosis patients and 173 healthy unrelated individuals by a method combining fluorescence labelling of PCR products and use of an automated DNA sequencer. Both patients and controls lived in the southwest of France (in the Pyrénées-Atlantiques) and had similar ethnic background. The distribution of the MOG haplotypes was not significantly different in the two groups (P = 0.38). This is not in favour of the implication of the MOG gene in the genetic component of multiple sclerosis, unless different independent mutations have occurred within this gene.

Monoclonal antibodies against Met-enkephalin as probe in the central nervous system.

Peptides with transmitter-like properties have been found in many brain areas. Immunochemical techniques have contributed most to clarification of the function and the pathway of these substances in neuronal systems. In this paper we report the production of 4 monoclonal antibodies against Met-enkephalin and their use in studying this peptide in rat cervical cord. Two of the antibodies recognize the COOH-terminus part of the Met-enkephalin, and do not cross-react with other known peptides. The other two antibodies are mainly directed against the NH2-terminus part of the peptide. Specific interactions of these monoclonal antibodies with regions of rat cervical cord were shown by immunochemistry techniques.

Analysis of HLA-A/B recombinant families with new polymorphic markers.

The MHC in humans is a much studied region of the genome, the genes of which display a high rate of polymorphism and a high rate of linkage disequilibrium. Four families in which intra-class-I recombination has occurred have been analyzed with six polymorphic markers between HLA-A and -B in order to determine the full haplotypes of the whole families and to localize the points of crossover. The previously proposed order of the markers was confirmed by recombination mapping. In one family, the crossover was shown to have occurred in the 20-kb stretch of DNA bounded by the two markers (P3B and P5) between which no evidence of linkage disequilibrium was found, a region which constitutes of only about 1% of the distance between HLA-A and HLA-B. Although supportive of the suggestion of a hot spot of recombination in this region, based on the apparent lack of linkage disequilibrium between the markers P3B and P5, more such families need to be tested in order to confirm or refute this hypothesis.

Three highly polymorphic microsatellites at the human myelin oligodendrocyte glycoprotein locus, 100 kb telomeric to HLA-F. Characterization and relation to HLA haplotypes.

The MOG locus, located on chromosomal bands 6p21.3-p22 and mapped about 100 kb telomeric to HLA-F, was isolated from cosmid ICRFc109A2434 and shown to contain three microsatellites. These CA-repeat polymorphic markers were characterized in a sample of 173 healthy unrelated individuals and 84 DNAs from the HLA Workshop reference panel, by a method combining fluorescence labeling of PCR products and use of an automated DNA sequencer. For the three markers, frequencies of heterozygotes are well predicted from allele frequencies by the Hardy-Weinberg rule, which suggests that problems of allele nonamplification are unlikely. Typing of cell lines homozygous in the HLA region allowed unambiguous definition of 81 HLA-MOG haplotypes and showed that several HLA ancestral haplotypes extended to the MOG region. The high degree of polymorphism (59%, 51%, and 81% at the three loci, respectively, and 87% at the haplotype level) makes these new markers informative for association or linkage studies with diseases such as hemochromatosis or multiple sclerosis, and for studies aimed at precisely delineating the site of crossover in chromosomes in which recombination occurred in the distal part of the HLA class I region.

New highly polymorphic microsatellite marker in linkage disequilibrium with HLA-B.

The difficulty of molecular typing of the HLA class I genes and the relevance of the genes of this region to disease susceptibility and transplantation have provided an impetus to develop useful typing markers. We have characterized by polymerase chain reaction analysis a new highly informative CA repeat localized approximately 25-kb centromeric to the gene HLA-B and 10-kb telomeric to the gene MICA. Twelve alleles defined by length were found in a sample of French Basques, with the PIC being 0.82. A detailed haplotype analysis was performed to investigate the association between this microsatellite and two others markers of the region (HLA-B gene and TNF region microsatellite). The 10 haplotypes with the highest estimated frequencies show evidence of a gametic association or linkage disequilibrium. A very strong association between the expressed HLA-B polymorphism and microsatellite alleles was also revealed in this sample and confirmed in the workshop cells lines of the Fourth Asia-Oceania Histocompatibility Workshop. This marker can be used in the fine mapping of this region and the association with some alleles of HLA-B may allow the replacement of HLA-B typing at least in a preliminary study. Moreover, these studies support the hypothesis of a high mutability for large alleles in microsatellite loci.

A susceptibility region for myasthenia gravis extending into the HLA-class I sector telomeric to HLA-C.

We have analyzed a series of HLA region markers in 207 UK Caucasoids with early-onset myasthenia gravis (EOMG, onset before age 40), where there is a strong female bias. The well known associations with HLA-DR3 and -B8 have now proved to be significantly stronger in the 165 females than in the 42 males. In patients (of either sex) lacking -DR3, there was also a significant increase in HLA-DR2. Although the muscle weakness in EOMG is clearly mediated by autoantibodies, the associations are consistently stronger with HLA-B8 (in class I) than with HLADR3 (in class II), as confirmed here. We therefore typed 87-137 cases for polymorphisms at four loci in the intervening class III region, and also at three in the adjacent stretch of class I. At each locus, one allele tended to co-occur with HLA-B8 and showed strong and highly significant associations in the patients. There appeared to be a region of maximal susceptibility extending from HSP70 (in class III) past HLA-B and HLA-C at least 600 kb telomerically into the class I region, which is now being mapped in detail. Any candidate genes here that act shortly after puberty may allow more precise localization of susceptibility.

A new immunological approach to the detection and the quantitation of the Met5-enkephalin precursors in rat brain.

We describe a new approach to the detection and quantitation of the enkephalin precursor. The approach is based on the production of antibody against a sequential determinant which is obtained specifically and quantitatively from the enkephalin precursor by tryptic hydrolysis. We chose the hexapeptide Tyr-Gly-Gly-Phe-Met-Arg and developed antibody against the C-terminus of this peptide. The hexapeptide is released from the precursor by mere tryptic cleavage. The antibody permits us to detect the Met-enkephalin precursor at the picomole level. Using this approach, we have quantitated the precursor forms in rat striatum and hypothalamus. The precursor/Met-enkephalin ratio was close to 3/4. This result was in very good agreement with the ratio determined previously in the bovine adrenal medulla.

Coexistence of putative neuroactive substances in lateral olivocochlear neurons of rat and guinea pig.

We have used the retrograde axonal transport of Fast Blue, injected intra-cochlearly, to identify in the rat lateral superior olive (LSO) neurons which belong to the lateral olivocochlear system (LOCS). Using immunohistofluorescence technique, we have localized within Fast Blue-labeled neurons immunostainings for enkephalins (Met-enkephalin, Met-enkephalin-Arg6-Gly7-Leu8), dynorphins (alpha-neo-endorphin, dynorphin 1-17) or choline acetyltransferase (ChAT). Many Fast Blue-labeled neurons did not show any immunostaining, but all the immunostained neurons found in the LSO were Fast Blue-labeled. In immunohistofluorescence colocalization experiments of two antigens, we could colocalize within the same neurons of the rat LSO immunostainings for ChAT and enkephalins and for ChAT and dynorphins. In each case, neurons only immunostained for ChAT, enkephalins or dynorphins could also be observed. A colocalization of the immunostainings for Met-enkephalin and dynorphins within neurons of the guinea pig and rat LSO was also found. However, in this case, neurons which did not show colocalization were only Met-enkephalin-immunoreactive, thus suggesting that all the dynorphins immunoreactive LSO neurons also contain enkephalins. These findings support the idea that the neurons of the LSO which contain ChAT-, enkephalin- or dynorphin-immunostainings project to the cochlea and belong to the LOCS. It can also be concluded that acetylcholine, enkephalins and dynorphins coexist within a same population of neurons of the LOCS, although other patterns of co-containment of neuroactive substances within LOCS neurons may also exist.

Compound Poisson approximation and testing for gene clusters with multigene families.

We present in this article a compound Poisson approximation for computing probabilities involved in significance tests for conserved genomic regions between different species. We consider the case when the conserved genomic regions are found by the reference region approach. An important aspect of our computations is the fact that we are taking into account the existence of multigene families. We obtain convergence results for the error of our approximation by using the Stein-Chen method for compound Poisson approximation.

Cross-reactivity of a monoclonal anti-[Met5]enkephalin antibody with cholecystokinin peptides.

The hypothesis of a conformational similarity between the unsulfated form of cholecystokinin C-terminal heptapeptide and [Met5]enkephalin was proposed by Schiller and colleagues. To test this possibility, we investigated the competition of cholecystokinin C-terminal peptides for the binding to a monoclonal anti-[Met5]enkephalin antibody. We observed that the antibody immunoprecipitated cholecystokinin peptides having at least the six C-terminal amino acids. Unlike the opiate receptors which had no affinity for the sulfated heptapeptide, the monoclonal antibody recognized the sulfated form of the C-terminal octapeptide of cholecystokinin.

B30.2-like domain proteins: a growing family.

The B30.2 domain is a conserved domain of around 170 amino acids. It is found associated with different protein domains: immunoglobulin domain in the case of butyrophilin and Ring Finger domain in the case of Ret Finger Protein. B30.2 should therefore be considered a migratory domain. We here report new members of these families as well as new protein families having the B30.2 domain, and we tentatively propose a general function for this domain.

The MHC big bang.

The human Major Histocompatibility Complex (MHC) shares similarities with three other chromosome regions in human. This could be the vestige of ancestral large scale duplications. We discuss here the possibility i) that these duplications occurred during two rounds of tetraploidization supposed to have taken place during chordate evolution before the jawed vertebrate radiation, and ii) that one of the quadruplicate regions, relaxed of functional constraints, gave rise to the vertebrate MHC by a quick round of gene cis-duplication and cis-exon shuffling. These different rounds of cis-duplications and exon shufflings allowed the emergence of new genes participating in novel biological functions i.e. adaptive immune responses. Cis-duplications and cis-exon shufflings are ongoing processes in the evolution of some of these genes in this region as they have occurred and were fixed at different times and in different lineages during vertebrate evolution. In contrast, other genes within the MHC have remained stable since the emergence of jawed vertebrates.

"Paleogenomics": looking in the past to the future.

The complete sequence of the human and other vertebrate and nonvertebrate genomes provide a wealth of information on the organization, relationships and evolution of the metazoans. Soon the fine structure of our innermost biological identity will be unveiled and what has so far remained deep and secret will shine like an unearthed treasure and shape and fuel our future quests. A key treasure, for many molecular scientists interested in molecular evolution and development would be the knowledge of the genome of the ancestral precursor of all metazoans. In the absence of fossil DNA, this knowledge will forever remain a yearning for dreamy molecular biologists. And yet, will not the power of deduction and reconstitution of information gained through man's sophisticated technologies one day recreate a "virtual" metazoan ancestor?