Stéphane Dréano

Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021

Sinorhizobium meliloti is an α-proteobacterium that forms agronomically important N2-fixing root nodules in legumes. We report here the complete sequence of the largest constituent of its genome, a 62.7% GC-rich 3,654,135-bp circular chromosome. Annotation allowed assignment of a function to 59% of the 3,341 predicted protein-coding ORFs, the rest exhibiting partial, weak, or no similarity with any known sequence. Unexpectedly, the level of reiteration within this replicon is low, with only two genes duplicated with more than 90% nucleotide sequence identity, transposon elements accounting for 2.2% of the sequence, and a few hundred short repeated palindromic motifs (RIME1, RIME2, and C) widespread over the chromosome. Three regions with a significantly lower GC content are most likely of external origin. Detailed annotation revealed that this replicon contains all housekeeping genes except two essential genes that are located on pSymB. Amino acid/peptide transport and degradation and sugar metabolism appear as two major features of the S. meliloti chromosome. The presence in this replicon of a large number of nucleotide cyclases with a peculiar structure, as well as of genes homologous to virulence determinants of animal and plant pathogens, opens perspectives in the study of this bacterium both as a free-living soil microorganism and as a plant symbiont.

Mapping and identification of essential gene functions on the X chromosome of Drosophila

The Drosophila melanogaster genome consists of four chromosomes that contain 165 Mb of DNA, 120 Mb of which are euchromatic. The two Drosophila Genome Projects, in collaboration with Celera Genomics Systems, have sequenced the genome, complementing the previously established physical and genetic maps. In addition, the Berkeley Drosophila Genome Project has undertaken large‐scale functional analysis based on mutagenesis by transposable P element insertions into autosomes. Here, we present a large‐scale P element insertion screen for vital gene functions and a BAC tiling map for the X chromosome. A collection of 501 X‐chromosomal P element insertion lines was used to map essential genes cytogenetically and to establish short sequence tags (STSs) linking the insertion sites to the genome. The distribution of the P element integration sites, the identified genes and transcription units as well as the expression patterns of the P‐element‐tagged enhancers is described and discussed.

Facilitating genome navigation: survey sequencing and dense radiation-hybrid gene mapping

Accurate and comprehensive sequence coverage for large genomes has been restricted to only a few species of specific interest. Lower sequence coverage (survey sequencing) of related species can yield a wealth of information about gene content and putative regulatory elements. But survey sequences lack long-range continuity and provide only a fragmented view of a genome. Here we show the usefulness of combining survey sequencing with dense radiation-hybrid (RH) maps for extracting maximum comparative genome information from model organisms. Based on results from the canine system, we propose that from now on all low-pass sequencing projects should be accompanied by a dense, gene-based RH map-construction effort to extract maximum information from the genome with a marginal extra cost.

Naturally occurring melanomas in dogs as models for non-UV pathways of human melanomas.

Spontaneously occurring melanomas are frequent in dogs. They appear at the same localizations as in humans, i.e. skin, mucosal sites, nail matrix and eyes. They display variable behaviors: tumors at oral localizations are more frequent and aggressive than at other anatomical sites. Interestingly, dog melanomas are associated with strong breed predispositions and overrepresentation of black‐coated dogs. Epidemiological analysis of 2350 affected dogs showed that poodles are at high risk of developing oral melanoma, while schnauzers or Beauce shepherds mostly developped cutaneous melanoma. Clinical and histopathological analyses were performed on a cohort of 153 cases with a 4‐yr follow‐up. Histopathological characterization showed that most canine tumors are intradermal and homologous to human rare morphological melanomas types – ‘nevocytoid type’ and ‘animal type’‐. Tumor cDNA sequencing data, obtained from 95 dogs for six genes, relevant to human melanoma classification, detected somatic mutations in oral melanoma, in NRAS and PTEN genes, at human hotspot sites, but not in BRAF. Altogether, these findings support the relevance of the dog model for comparative oncology of melanomas, especially for the elucidation of non‐UV induced pathways.

A 356-Kb Sequence of the Subtelomeric Part of the MHC Class I Region

The subtelomeric part of the MHC Class I region contains 11 of the 21 genes described on chromosome 6 at position 6p21.3. The general organization of those and other genes resident in the region was revealed by determining a 356,376 bp sequence. Potential exons for new genes were identified by computer analysis and a large number of ESTs were selected by testing the sequence by the BLAST algorithm against the GenBank nonredundant and EST databases. Most of the ESTs are clustered in two regions. In contrast, the whole HLA-gene region is crammed with LINE and SINE repeats, fragments of genes and microsatellites, which tends to hinder the identification of new genes.

Ancestral T-Box Mutation Is Present in Many, but Not All, Short-Tailed Dog Breeds

Dogs differ greatly in their morphological characteristics including various tail phenotypes. Congenitally short-tailed dogs are present in many breeds; however, the causative mutation located in the T-box transcription factor T gene (C189G) had only been described in the bobtailed Pembroke Welsh Corgis. We investigated here the presence of the T gene mutation in 23 other breeds (360 dogs, including 156 natural short tailed) in which natural bobtailed dogs exist. In the 17 breeds in which the C189G mutation was observed, there was a perfect correlation between this mutation and the short-tail phenotype. However, 6 breeds did not carry the known substitution or any other mutations in the T gene coding regions. No dogs were found to be homozygous for the C189G mutation, suggesting that the homozygous condition is lethal. In order to study the effect of the T gene mutation on litter size, we compared the number of puppies born from short-tailed parents to that born from long-tailed parents. In the Swedish Vallhund breed, we observed a 29% decrease in the litter size when both parents were short tailed. Given that the T gene mutation is not present in all breeds of short-tailed dog, there must be yet other genetic factors affecting tail phenotypes to be discovered.

Paraffin-embedded tissue is less accurate than frozen section analysis for determining VHL mutational status in sporadic renal cell carcinoma

INTRODUCTION Literature controversies exist regarding the prognostic value of VHL mutations. The objective was to compare paraffin-embedded and frozen section specimens for VHL mutations detection and to evaluate the reliability of DNA analysis in formalin-fixed tissues. METHODS Seventy-six patients with clear cell renal cell carcinoma (RCC) previously assessed for VHL status from frozen samples were included. Seventy-three tumor samples were known to be mutated for VHL. DNA was extracted and an electrophoresis was performed to determine DNA quality. The whole coding sequence was synthesized by double PCR amplification followed by sequencing. Sequencing results were compared with those previously determined from frozen samples. RESULTS DNA could be extracted from the 76 paraffin samples. DNA quality was highly degraded and significantly less amplified by PCR in 34.2%, resulting in no sequence available for analysis in 57.7% and discordance with frozen samples in 42.3% of the cases respectively. VHL mutations were found in 52.1% of the whole paraffin samples whereas 98% were mutated; 72% could be sequenced, resulting in 69.1% of VHL mutations in this subset. Only half of observed mutations were fully consistent with frozen analysis in the 3 exons. Neomutations were found in 10.5% and 28.9% of known mutations in frozen samples were not detected in paraffin blocks. Only DNA quality significantly influenced PCR amplification and sequencing. CONCLUSION Tumoral DNA extraction and VHL mutation analysis can be performed from formalin-fixed paraffin-embedded (FFPE) tissue in RCC. But mutations identified tissues are not strictly concordant with those from frozen analysis and therefore results obtained from FFPE samples should be interpreted with care.

Aggregation dynamics and identification of aggregation-prone mutants of the von Hippel–Lindau tumor suppressor protein

ABSTRACT Quality control mechanisms promote aggregation and degradation of misfolded proteins. In budding yeast, the human von Hippel–Lindau protein (pVHL, officially known as VHL) is misfolded and forms aggregates. Here, we investigated the aggregation of three pVHL isoforms (pVHL213, pVHL160, pVHL172) in fission yeast. The full-length pVHL213 isoform aggregates in highly dynamic small puncta and in large spherical inclusions, either close to the nucleus or to the cell ends. The large inclusions contain the yeast Hsp104 chaperone. Aggregate clearance is regulated by proteasomal degradation. The pVHL160 isoform forms dense foci and large irregularly shaped aggregates. In silico, prediction of pVHL aggregation propensity identified a key aggregation-promoting region within exon 2. Consistently, the pVHL172 isoform, which lacks exon 2, formed rare reduced inclusions. We studied the aggregation propensity of pVHL variants harbouring missense mutations found in kidney carcinomas. We show that the P86L mutation stimulated small aggregate formation, the P146A mutation increased large inclusion formation, whereas the I151S mutant destabilized pVHL. The prefoldin subunit Pac10 (the human homolog VBP-1 binds to pVHL) is required for pVHL stability. Reduction of soluble functional pVHL might be crucial in VHL-related diseases. Highlighted Article: If misfolded, the von Hippel–Lindau (pVHL) tumor suppressor protein is prone to aggregate and to be degraded. Tumorigenic mutations might compromise its folding. We identify new factors regulating pVHL folding.

Nebria Species (Subfamily Nebriinae, Family: Carabidae) from the Romanian Carpathians. Morphological and Molecular Data

Abstract Among the ten species of the Nebria genus, present in the Romanian Carpathians, seven are the object of morphological, geographical distribution and molecular biological studies: Nebria (Eunebria) jockischi hoepfneri Dejean, 1826, Nebria (Boreonebria) heegeri Dejean, 1826, Nebria (Boreonebria) gyllenhali Schönherr, 1806, Nebria (Alpaeonebria) reichei Dejean, 1826, Nebria (Alpaeonebria) reitteri Rybinsky, 1902, Nebria (Alpaeonebria) bissenica Bielz, 1887, Nebria (Nebria) transsylvanica Germar, 1824, Nebria (Nebria) femoralis alpigrada Csiki 1905), collected from the Maramureș Mt., Rodnei Mt., Parâng Mt., Rarău Mt., Făgăraș Mt., Cozia Mt., Bucegi Mt., Retezat Mt., Muntele Mic and Semenic Mt. The morphological description exploits the body size, the elytral reflection, the colour of the appendages and the legs, the shape of the first antennary segment, its chaetotaxy and that of the submentum, the shape and size of the elytra and alae, the position of the bristles on the ventrites 4-5-6. The shape of the first antennary segment and its chaetotaxy appear as more discriminating criteria, but supposes the integrity of the bristles. The identification is sometimes malaise due to the fragility of the bristles (first antennomere, submentum). Nebria (Alpaeonebria) reichei Dejean, 1826 has a variable chaetotaxy of antenna, 1 to 3 bristles on the first antennomere. The individuals with yellow appendages and legs provided with 2 unequal length can be confused with transsylvanica. They are distinguished by the triangular shape of the aileron (S-shaped in transsylvanica). Molecular data are given for the first time on Carpathian Nebria. The mitochondrial markers (COI I, cyt b) clearly identify the species studied and confirm that alpigrada does not belong to transsylvanica. The results show an infraspecific variability of geographic and altitudinal origin in jockischi one of the most widespread species (gyllenhali, jockischi, reichei).