A physical map of the heterozygous grapevine 'Cabernet Sauvignon' allows mapping candidate genes for disease resistance

<p>Abstract</p> <p>Background</p> <p>Whole-genome physical maps facilitate genome sequencing, sequence assembly, mapping of candidate genes, and the design of targeted genetic markers. An automated protocol was used to construct a <it>Vitis vinifera </it>'Cabernet Sauvignon' physical map. The quality of the result was addressed with regard to the effect of high heterozygosity on the accuracy of contig assembly. Its usefulness for the genome-wide mapping of genes for disease resistance, which is an important trait for grapevine, was then assessed.</p> <p>Results</p> <p>The physical map included 29,727 BAC clones assembled into 1,770 contigs, spanning 715,684 kbp, and corresponding to 1.5-fold the genome size. Map inflation was due to high heterozygosity, which caused either the separation of allelic BACs in two different contigs, or local mis-assembly in contigs containing BACs from the two haplotypes. Genetic markers anchored 395 contigs or 255,476 kbp to chromosomes. The fully automated assembly and anchorage procedures were validated by BAC-by-BAC blast of the end sequences against the grape genome sequence, unveiling 7.3% of chimerical contigs. The distribution across the physical map of candidate genes for non-host and host resistance, and for defence signalling pathways was then studied. NBS-LRR and RLK genes for host resistance were found in 424 contigs, 133 of them (32%) were assigned to chromosomes, on which they are mostly organised in clusters. Non-host and defence signalling genes were found in 99 contigs dispersed without a discernable pattern across the genome.</p> <p>Conclusion</p> <p>Despite some limitations that interfere with the correct assembly of heterozygous clones into contigs, the 'Cabernet Sauvignon' physical map is a useful and reliable intermediary step between a genetic map and the genome sequence. This tool was successfully exploited for a quick mapping of complex families of genes, and it strengthened previous clues of co-localisation of major NBS-LRR clusters and disease resistance <it>loci </it>in grapevine.</p

Genetic dissection of the type VI secretion system in Acinetobacter and identification of a novel peptidoglycan hydrolase, TagX, required for its biogenesis

The type VI secretion system (T6SS) is a widespread secretory apparatus produced by Gram-negative bacteria that has emerged as a potent mediator of antibacterial activity during interbacterial interactions. Most Acinetobacter species produce a genetically conserved T6SS, although the expression and functionality of this system vary among different strains. Some pathogenic Acinetobacter baumannii strains activate this secretion system via the spontaneous loss of a plasmid carrying T6SS repressors. In this work, we compared the expression of T6SS-related genes via transcriptome sequencing and differential proteomics in cells with and without the plasmid. This approach, together with the mutational analysis of the T6SS clusters, led to the determination of the genetic components required to elaborate a functional T6SS in the nosocomial pathogen A. baumannii and the nonpathogen A. baylyi. By constructing a comprehensive combination of mutants with changes in the T6SS-associated vgrG genes, we delineated their relative contributions to T6SS function. We further determined the importance of two effectors, including an effector-immunity pair, for antibacterial activity. Our genetic analysis led to the identification of an essential membrane-associated structural component named TagX, which we have characterized as a peptidoglycan hydrolase possessing l,d-endopeptidase activity. TagX shows homology to known bacteriophage l,d-endopeptidases and is conserved in the T6SS clusters of several bacterial species. We propose that TagX is the first identified enzyme that fulfills the important role of enabling the transit of T6SS machinery across the peptidoglycan layer of the T6SS-producing bacterium

Numerous Novel Annotations of the Human Genome Sequence Supported by a 5′-End–Enriched cDNA Collection

A collection of 90,000 human cDNA clones generated to increase the fraction of “full-length” cDNAs available was analyzed by sequence alignment on the human genome assembly. Five hundred fifty-two gene models not found in LocusLink, with coding regions of at least 300 bp, were defined by using this collection. Exon composition proposed for novel genes showed an average of 4.7 exons per gene. In 20% of the cases, at least half of the exons predicted for new genes coincided with evolutionary conserved regions defined by sequence comparisons with the pufferfish Tetraodon nigroviridis. Among this subset, CpG islands were observed at the 5′ end of 75%. In-frame stop codons upstream of the initiator ATG were present in 49% of the new genes, and 16% contained a coding region comprising at least 50% of the cDNA sequence. This cDNA resource also provided candidate small protein-coding genes, usually not included in genome annotations. In addition, analysis of a sample from this cDNA collection indicates that ∼380 gene models described in LocusLink could be extended at their 5′ end by at least one new exon. Finally, this cDNA resource provided an experimental support for annotations based exclusively on predictions, thus representing a resource substantially improving the human genome annotation

A refined picture of the native Amine Dehydrogenase family revealed by extensive biodiversity screening

Native amine dehydrogenases offer sustainable access to chiral amines, so the search for scaffolds capable of converting more diverse carbonyl compounds is required to reach the full potential of this alternative to conventional synthetic reductive aminations. Here we report a multidisciplinary strategy combining bioinformatics, chemoinformatics and biocatalysis to extensively screen billions of sequences in silico and to efficiently find native amine dehydrogenases features using computational approaches. In this way, we achieve a comprehensive overview of the initial native amine dehydrogenase family, extending it from 2,011 to 17,959 sequences, and identify native amine dehydrogenases with non-reported substrate spectra, including hindered carbonyls and ethyl ketones, and accepting methylamine and cyclopropylamine as amine donor. We also present preliminary model-based structural information to inform the design of potential (R)-selective amine dehydrogenases, as native amine dehydrogenases are mostly (S)-selective. This integrated strategy paves the way for expanding the resource of other enzyme families and in highlighting enzymes with original features. INTRODUCTIO

A family of Native Amine Dehydrogenases for the Asymmetric Reductive Amination of Ketones

The asymmetric reductive amination of ketones enables the one-step synthesis of chiral amines from readily available starting materials. Here we report the discovery of a family of native NAD(P)H-dependent Amine Dehydrogenases (nat-AmDHs) competent for the asymmetric reductive amination of aliphatic and alicyclic ketones, adding significantly to the biocatalytic toolbox available for chiral amine synthesis. Studies of ketone and amine substrate specificity and kinetics reveal a strong preference for aliphatic ketones and aldehydes, with activities of up to 614.5 mU mg-1 for cyclohexanone with ammonia and 851.3 mU mg-1 for isobutyraldehyde with methylamine as amine donor. Crystal structures of three nat-AmDHs (AmDH4, MsmeAmDH and CfusAmDH) reveal the active site determinants of substrate and cofactor specificity and enable the rational engineering of AmDH4 for generated activity towards pentan-2-one. Analysis of the 3D-catalytic site distribution among bacterial biodiversity revealed a superfamily of divergent proteins with representative specificities ranging from amino acid substrates to hydrophobic ketones

Physical mapping and re-sequencing of a set of resistance related genes in grapevine

Physical mapping and re-sequencing of a set of resistance related genes in grapevine. 17. Conference Plant and Animal Genome

Single-cell genomics of multiple uncultured stramenopiles reveals underestimated functional diversity across oceans

Single-celled eukaryotes (protists) are critical players in global biogeochemical cycling of nutrients and energy in the oceans. While their roles as primary producers and grazers are well appreciated, other aspects of their life histories remain obscure due to challenges in culturing and sequencing their natural diversity. Here, we exploit single-cell genomics and metagenomics data from the circumglobal Tara Oceans expedition to analyze the genome content and apparent oceanic distribution of seven prevalent lineages of uncultured heterotrophic stramenopiles. Based on the available data, each sequenced genome or genotype appears to have a specific oceanic distribution, principally correlated with water temperature and depth. The genome content provides hypotheses for specialization in terms of cell motility, food spectra, and trophic stages, including the potential impact on their lifestyles of horizontal gene transfer from prokaryotes. Our results support the idea that prominent heterotrophic marine protists perform diverse functions in ocean ecology