Construction of a cytogenetically anchored microsatellite map in rabbit.

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.

A first generation bovine BAC-based physical map.

A first generation clone-based physical map for the bovine genome was constructed combining, fluorescent double digestion fingerprinting and sequence tagged site (STS) marker screening. The BAC clones were selected from an Inra BAC library (105,984 clones) and a part of the CHORI-240 BAC library (26,500 clones). The contigs were anchored using the screening information for a total of 1303 markers (451 microsatellites, 471 genes, 127 EST, and 254 BAC ends). The final map, which consists of 6615 contigs assembled from 100,923 clones, will be a valuable tool for genomic research in ruminants, including targeted marker production, positional cloning or targeted sequencing of regions of specific interest.

Putative FLJ20436 gene characterisation in goat. Observed ubiquitous expression in goat and transgenic mice allowed to restrict the location of an hypothesised insulator element.

Eukaryotic genomes are organised into independent domains through the establishment of boundaries which allow to have distinct pattern of gene expression both during development and in differentiated cells. The previously reported site independent expression of the mammary-specific goat alpha-lactalbumin gene in transgenic mice suggested the existence of cis-regulatory elements located upstream of this gene. The nearby presence of a second ubiquitously expressed gene (the cyclin T1 gene) allowed to define two chromatin domains putatively separated by a boundary insulator-like element. In this study, the characterisation of a third putative gene (FLJ20436) present between the alpha-lactalbumin and the cyclin T1 loci is reported. It was found to be a functional gene, ubiquitously expressed both in goat and transgenic mice. A complex pattern of alternative splicing events was observed in several analysed tissues leading to various mRNAs and putative FLJ20436 proteins, as suggested in human and mouse species. This allowed us to assign this gene to one of the two hypothesised chromatin domains, refining the location of the potential insulator element.

The genome sequence of the entomopathogenic bacterium Photorhabdus luminescens.

Photorhabdus luminescens is a symbiont of nematodes and a broad-spectrum insect pathogen. The complete genome sequence of strain TT01 is 5,688,987 base pairs (bp) long and contains 4,839 predicted protein-coding genes. Strikingly, it encodes a large number of adhesins, toxins, hemolysins, proteases and lipases, and contains a wide array of antibiotic synthesizing genes. These proteins are likely to play a role in the elimination of competitors, host colonization, invasion and bioconversion of the insect cadaver, making P. luminescens a promising model for the study of symbiosis and host-pathogen interactions. Comparison with the genomes of related bacteria reveals the acquisition of virulence factors by extensive horizontal transfer and provides clues about the evolution of an insect pathogen. Moreover, newly identified insecticidal proteins may be effective alternatives for the control of insect pests.

A mutation in the LAMC2 gene causes the Herlitz junctional epidermolysis bullosa (H-JEB) in two French draft horse breeds.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterised by skin blistering and fragility. In humans, one of the most severe forms of EB known as Herlitz-junctional EB (H-JEB), is caused by mutations in the laminin 5 genes. EB has been described in several species, like cattle, sheep, dogs, cats and horses where the mutation, a cytosine insertion in exon 10 of the LAMC2 gene, was very recently identified in Belgian horses as the mutation responsible for JEB. In this study, the same mutation was found to be totally associated with the JEB phenotype in two French draft horse breeds, Trait Breton and Trait Comtois. This result provides breeders a molecular test to better manage their breeding strategies by genetic counselling.

A mutation in the MATP gene causes the cream coat colour in the horse.

In horses, basic colours such as bay or chestnut may be partially diluted to buckskin and palomino, or extremely diluted to cream, a nearly white colour with pink skin and blue eyes. This dilution is expected to be controlled by one gene and we used both candidate gene and positional cloning strategies to identify the "cream mutation". A horse panel including reference colours was established and typed for different markers within or in the neighbourhood of two candidate genes. Our data suggest that the causal mutation, a G to A transition, is localised in exon 2 of the MATP gene leading to an aspartic acid to asparagine substitution in the encoded protein. This conserved mutation was also described in mice and humans, but not in medaka.

Sequence of goat cyclin T1 cDNA, gene organisation and expression analysis.

The cyclin T1 (Cyc T1) protein has been recently identified, associated with the cyclin-dependent kinase 9 (CDK 9), as to be involved in the transcriptional activation of the Human Immunodeficiency Virus type 1 (HIV-1) by the Tat protein. In this study, the sequence of the 7 kb goat Cyc T1 cDNA is reported as well as the exon/intron structure of the gene. Its observed ubiquitous expression is consistent with the promoter structure.

Sheep/human comparative map in a chromosome region involved in scrapie incubation time shows multiple breakpoints between human chromosomes 14 and 15 and sheep chromosomes 7 and 18.

A chromosome region involved in scrapie incubation time was identified on sheep chromosome 18 (OAR18). Since OAR18 (and OAR7) share conserved chromosome segments with human chromosomes HSA14 and HSA15, a dense map of type I markers was constructed by FISH mapping of bacterial artificial chromosomes containing genes located on these human chromosomes. In this study, we used the complete human sequence information (gene positions in megabases, Mb) to locate approximately one gene every 2 Mb on HSA15 (19 genes mapped between 19.51 and 66.02 Mb) and on HSA14 (11 genes between 73.24 and 102.62 Mb). Combined with previous work carried out in cattle and goats, our results made it possible to refine the comparative map between ruminants and humans for these two highly rearranged chromosomes (10 segments on HSA15 and 7 on HSA14). Furthermore, we identified relatively short intervals containing evolutionary breakpoints, which is a prerequisite to position them precisely. This work is also the first step in the cloning of the region involved in scrapie incubation period in sheep.

Sequence determination and expression of the ovine doppel-encoding gene in transgenic mice.

The ovine Doppel-encoding gene transcription unit (TU) and proximal flanking regions were cloned from a bacterial artificial chromosome (BAC) and sequenced. The 4586 bp TU is composed of two exons and one intron. When compared with its human counterpart, beside the open reading frame and part of the 3' untranslated sequence, significant regions of homology were found within the intron and the proximal 5' flanking regions. Sequence analysis of the BAC clone revealed that the ovine Prnd gene is located around 52 kb downstream of the Prnp locus. Expression of the sheep and murine Prnd genes was observed in all tissues analyzed of transgenic mice bearing the BAC insert, with a noticeable highest expression level observed in the testis, with no associated noticeable phenotype. The present data suggest that, in contrast to ovine Prnp, ovine Prnd expression in transgenic mice has no obvious influence on conferred susceptibility to scrapie.

Characterization of transposon Tn1549, conferring VanB-type resistance in Enterococcus spp.

Transfer of VanB-type resistance to glycopeptides among enterococci has been reported to be associated with the movement of large chromosomal genetic elements or of plasmids. The authors report the characterization of the 34 kb transposon Tn1549 borne by a plasmid related to pAD1 and conferring vancomycin resistance in clinical isolates of Enterococcus spp. Tn1549 contained 30 ORFs and appeared to be organized like the Tn916 family of conjugative transposons into three functional regions: (i) the right end, implicated in the excision-integration process; (ii) the central part, in which the vanB2 operon replaces the tet(M) gene; and (iii) the left extremity, in which eight of the 18 ORFs could be implicated in the conjugative transfer.