François Piumi

Genome sequence of the model mushroom Schizophyllum commune

Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the speciess unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Cytogenetic localization of 136 genes in the horse: comparative mapping with the human genome

The aim of this study was to increase the number of type I markers on the horse cytogenetic map and to improve comparison with maps of other species, thus facilitating positional candidate cloning studies. BAC clones from two different sources were FISH mapped: homologous horse BAC clones selected from our newly extended BAC library using consensus primer sequences and heterologous goat BAC clones. We report the localization of 136 genes on the horse cytogenetic map, almost doubling the number of cytogenetically mapped genes with 48 localizations from horse BAC clones and 88 from goat BAC clones. For the first time, genes were mapped to ECA13p, ECA29, and probably ECA30. A total of 284 genes are now FISH mapped on the horse chromosomes. Comparison with the human map defines 113 conserved segments that include new homologous segments not identified by Zoo-FISH on ECA7 and ECA13p.

FOLy: An integrated database for the classification and functional annotation of fungal oxidoreductases potentially involved in the degradation of lignin and related aromatic compounds

The breakdown of lignin by fungi is a key step during carbon recycling in terrestrial ecosystems. This process is of great interest for green and white biotechnological applications. Given the importance of these enzymatic processes, we have classified the enzymes potentially involved in lignin catabolism into sequence-based families and integrated them in a newly developed database, designated Fungal Oxidative Lignin enzymes (FOLy). Families were defined after sequence similarity searches starting from protein sequences and validated by the convergence of results with biochemical experiments reported in the literature. The resulting database was applied as a tool for the functional annotation of genomes from different fungi, namely (i) the Basidiomycota Coprinopsis cinerea, Phanerochaete chrysosporium and Ustilago maydis and (ii) the Ascomycota Aspergillus nidulans and Trichoderma reesei. Genomic comparison of the oxidoreductases of these fungi revealed significant differences in the putative enzyme arsenals. Two Ascomycota fungal genomes were annotated and new candidate genes were identified that could be useful for lignin degradation and (or) melanin synthesis, and their function investigated experimentally. This database efforts aims at providing the means to get new insights for the understanding and biotechnological exploitation of the lignin degradation. A WWW server giving access to the routinely updated FOLy classifications of enzymes potentially involved in lignin degradation can be found at http://foly.esil.univ-mrs.fr.

Construction of a 5000 rad whole-genome radiation hybrid panel in the horse and generation of a comprehensive and comparative map for ECA11

Abstract. A 5000rad whole-genome radiation hybrid (RH) panel was created for the horse. The usefulness of the panel for generating physically ordered maps of individual equine chromosomes was tested by typing 24 markers on horse Chromosome 11 (ECA11). The overall retention of markers on this chromosome was 43.6%. Almost complete retention of two of the typed markers—CA062 and AHT44—clearly indicated the location of thymidine kinase gene on the short arm of ECA11. Seven of the typed markers were FISH mapped to align the RH and cytogenetic maps. With the RH-MAPPER approach, a physically ordered map comprising four linkage groups and incorporating all the markers was obtained. The study provides the first comprehensive map for a horse chromosome that integrates all available mapping data and adds new information that spans the entire length of the equine chromosome. The map clearly underlines the resolving power and utility of the panel and emphasizes the need to have uniformly distributed cytogenetic markers for appropriate alignment of RH map with the chromosome. A comparative status of the ECA11 map in relation to the corresponding human/mouse chromosome is presented.

Hepatic gene expression profiles in juvenile rainbow trout (Oncorhynchus mykiss) fed fishmeal or fish oil-free diets

Reducing the reliance on fishery by-products as amino acid and fatty acid sources in feeds for farmed fish is a major objective today. We evaluated the effect of dietary fish oil or dietary fishmeal replacement by vegetable oils and plant proteins respectively through analysis of hepatic transcriptomes in rainbow trout (Oncorhynchus mykiss). Fish were fed right from first feeding with diets based on plant by-products before being killed. We analysed the hepatic gene profile using trout cDNA microarrays (9K). Our data showed that seventy-one and seventy-five genes were affected after fish oil and fishmeal replacement respectively. The major part of modified gene expression coding for proteins of the metabolic pathways was as follows: (i) a lower level of expression for genes of energy metabolism found in fish after fishmeal and fish oil replacement; (ii) a lower level of gene expression for fatty acid metabolism (biosynthesis) in fish fed with vegetable oils; (iii) a differential expression of actors of detoxification metabolism in trout fed with vegetable oils; (iv) a lower level of expression of genes involved in protein metabolism in fish fed with plant proteins. Overall, our data suggest that dietary fish oil replacement is linked to a decreased capacity of fatty acid biosynthesis (fatty acid synthase) and variation of detoxification metabolism (cytochrome P450s) whereas dietary fishmeal replacement may depress protein metabolism in the liver as reflected by glutamine synthetase.

Construction of a rabbit bacterial artificial chromosome (BAC) library: application to the mapping of the major histocompatibility complex to position 12q1.1

In the past five years, large DNA fragment libraries in Bacterial Artificial Chromosomes (BAC) (Shizuya et al. 1992) have permitted remarkable advances in the analysis of major domestic species of agronomic interest. Thus, BAC libraries representing from 3 to 10 haploid genome coverage are now available for a number of domestic animals. The success of the BAC libraries is mainly owing to the DNA fidelity of the genomic inserts and its low level of chimerism. In addition, BAC clones are amenable to readily build physical maps representing the whole genome and direct sequencing for efficient chromosome walking and large-scale sequencing projects. The rabbit ( Oryctolagus cuniculus ) genome has been poorly studied thus far, despite the use of this species in multiple physiological and immunological studies as well as transgenic experiments. Furthermore, a number of laboratories and private companies are interested in improving production as well as reproductive performances in rabbit, which requires the development of molecular tools. In this context, a BAC library would be of great value. Since no rabbit BAC library has yet been made available, to our knowledge, we constructed a library that currently represents three haploid genome equivalents. Despite a limited genome coverage, this kind of library has already proved its efficiency to start working on infrequently studied species. In farm animals, cytogenetic, physical, and comparative maps were developed with 1.5 and 3 genome equivalent BAC libraries for horse and ruminants, respectively (Mariat et al. 2000; Schibler et al. 1998). High-molecular-weight DNA was prepared from white blood cells isolated from a New Zealand sire rabbit which was homozygous DRAdd-DQAbb (Han et al. 1992) for the class II region of the rabbit RLA major histocompatibility complex (MHC). The procedure to construct the BAC library in the pBeloBAC11 vector by using theHindIII cloning site was identical to the one used to build our pig BAC library (Rogel-Gaillard et al. 1999). The whole BAC library comprised 84,480 clones with an average insert size of 100–110 kilobases (kb; Fig. 1), representing a theoretical threefold coverage of the haploid genome. The BAC library was stored in 96-well microtiter plates and was organized in a threedimension pooling system to prepare DNA ready to use for a PCR screening. Each of the 44 superpools corresponds to 20 plates of 96 clones, that is, a total of 1920 clones. The recovery of clones for a specific primer pair requires a two-step process. The first step corresponds to the screening of 44 superpool DNAs and three controls, that is, 47 PCRs, while the second step corresponds to the screening of the pools for the rows, columns, and plates, that is, 44 PCRs with controls per positive superpool. The library was screened upon request from different European and one American laboratory with primer pairs specific for 46 distinct sequences. Thirty-seven out of the 46 primer sets yielded between 1 and 10 clones with an average of 3.3 clones. No clone was recovered for the remaining 9 primer sets. In addition, the library was also screened for genes from each of the three MHC class I, II and III regions (Table 1). Primers for genes of the class I region were derived from the RLA classical class I R19 gene (Marche et al. 1985) and from a single-copy gene corresponding to R27 (Rebiere et al. 1987). Primers for genes of the RLA class II region were derived from the rabbit DRA, DRB, DQA, DQB, DMA, DMB, DPA, DPB genes and the human TAP1 gene (Table 1). Finally, the screening for class III genes was performed by using primers derived from the rabbit TNFAgene, the pigHSP70 gene, and the human TNX and NOTCH4 genes (Table 1). All the PCR products obtained with the different primer sets were sequenced, and nucleic acid similarity searches were conducted with a BLASTN program (Altschul et al., 1997) to assess the specificity of the sequences. The rabbit nucleotide sequences for NOTCH4, TNX, and TAP1 genes were submitted to EMBL/GenBank database and were given the accession numbers AJ297382, AJ297381, and AJ297383, respectively. Two distinct sequences were obtained Correspondence to:C. Rogel-Gaillard; E-mail: rogel@biotec.jouy. inra.fr Fig. 1. Mammalian Genome 12, 253–255 (2001). DOI: 10.1007/s003350010260

Comparative cytogenetic mapping reveals chromosome rearrangements between the X chromosomes of two closely related mammalian species (cattle and goats)

Cytogenetic localization of 24 BACs containing type I (genes and ESTs) and type II (microsatellites) markers were used to construct cytogenetic maps of caprine (CHI) and bovine (BTA) X chromosomes. Comparison of these two maps revealed that the distal region of the goat X long arm (CHI Xq38→q42) was located inside the bovine X chromosome, between PGK1 (BTA Xq25) and DVEPC137 (BTA Xq12). The marker order was globally conserved without any pericentric inversion, as previously postulated in the literature. The caprine centromere was found between DVEPC053 and DVEPC102 (belonging to the same band in the bovine X: BTA Xq41), whereas the bovine centromere was between DVEPC076 and DVEPC132, belonging to the same region of the caprine X chromosome (CHI Xq31→q33). The pseudoautosomal region was situated at the tip of the bovine X long arm and on the tiny short arm of the caprine X chromosome. In the non-pseudoautosomal (NPA) region, the synteny of coding sequences was well conserved between the human species and the two ruminant species, but the gene order was dramatically divergent. It is suggested that the 24 BACs of this study could constitute a new tool to measure phylogenetic distances between different mammalian species by comparing chromosome rearrangements inside the NPA region of the X.

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims:  Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector.

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.

Hypoxia-activated genes from early placenta are elevated in Preeclampsia, but not in Intra-Uterine Growth Retardation

BackgroundAs a first step to explore the possible relationships existing between the effects of low oxygen pressure in the first trimester placenta and placental pathologies developing from mid-gestation, two subtracted libraries totaling 2304 cDNA clones were constructed. For achieving this, two reciprocal suppressive/subtractive hybridization procedures (SSH) were applied to early (11 weeks) human placental villi after incubation either in normoxic or in hypoxic conditions. The clones from both libraries (1440 hypoxia-specific and 864 normoxia-specific) were spotted on nylon macroarrays. Complex cDNAs probes prepared from placental villi (either from early pregnancy, after hypoxic or normoxic culture conditions, or near term for controls or pathological placentas) were hybridized to the membranes.ResultsThree hundred and fifty nine clones presenting a hybridization signal above the background were sequenced and shown to correspond to 276 different genes. Nine of these genes are mitochondrial, while 267 are nuclear. Specific expression profiles characteristic of preeclampsia (PE) could be identified, as well as profiles specific of intra-uterine growth retardation (IUGR).Focusing on the chromosomal distribution of the fraction of genes that responded in at least one hybridization experiment, we could observe a highly significant chromosomal clustering of 54 genes into 8 chromosomal regions, four of which containing imprinted genes. Comparative mapping data indicate that these imprinted clusters are maintained in synteny in mice, and apparently in cattle and pigs, suggesting that the maintenance of such syntenies is requested for achieving a normal placental physiology in eutherian mammals.ConclusionWe could demonstrate that genes induced in PE were also genes highly expressed under hypoxic conditions (P = 5.10-5), which was not the case for isolated IUGR. Highly expressed placental genes may be in syntenies conserved interspecifically, suggesting that the maintenance of such clusters is requested for achieving a normal placental physiology in eutherian mammals.