Construction and characterization of a BAC library for functional genomics in Xenopus tropicalis.

Large insert genomic DNA libraries are useful resources for genomic studies. Although the genome of Xenopus tropicalis stands as the amphibian reference genome because it benefitted from large-scale sequencing studies, physical mapping resources such as BAC libraries are lagging behind. Here we present the construction and characterization of a BAC library that covers the whole X. tropicalis genome. We prepared this BAC library from the genomic DNA of X. tropicalis females of the Adiopodoume strain. We characterized BAC clones by screening for specific loci, by chromosomal localization using FISH and by systematic BAC end sequencing. The median insert size is about 110kbp and the library coverage is around six genome equivalents. We obtained a total of 163,787 BAC end sequences with mate pairs for 77,711 BAC clones. We mapped all BAC end sequences to the reference X. tropicalis genome assembly to enable the identification of BAC clones covering specific loci. Overall, this BAC library resource complements the knowledge of the X. tropicalis genome and should further promote its use as a reference genome for developmental biology studies and amphibian comparative genomics.

A functional genomic approach to the study of the milking ability in dairy sheep.

To understand the mechanisms underlying milk ability and more precisely the kinetics of milk emission, we compared teat transcriptome profiles from Lacaune ewes in the tails of the milk flow phenotypic distribution. Two different arrays containing respectively 1896 and 13 168 PCR products selected from several tissue-specific cDNA libraries, including mammary gland, allowed the identification of 73 differentially expressed genes between teats from high and low milk flow ewes. Genes involved in muscle contraction were identified as over-expressed, and genes encoding collagen were found to be under-expressed in teats from low milk flow ewes. We confirmed this underexpression of COL1A1 and COL1A2 in low-milk flow ewes using RT-qPCR. These results suggest that milking ability may be due to the capacity of the teat sphincter to relax during mechanical milking. We propose that an optimal condition for mechanical milking may require proper relaxation of the teats. To our knowledge, this is the first transcriptomic analysis studying milking ability, using udder tissue for gene expression profiling, which demonstrates that mechanical milking ability is not only determined by morphological features but also by tissue composition.

Terminal differentiation of goat mammary tissue during pregnancy requires the expression of genes involved in immune functions.

Terminal differentiation of mammary tissue into a functional epithelium that synthesizes and secretes milk occurs during pregnancy. The molecular mechanisms underlying this complex process are poorly understood, especially in ruminants. To obtain an overview of the ruminant mammary gland's final differentiation process, we conducted time-course gene expression analysis of five physiological stages: four during pregnancy (P46, P70, P90, and P110) and one after 40 days of lactation (L40). An appropriate loop experimental design was used to follow gene expression profiles. Using three nulliparous (pregnancy) or primiparous (lactation) goats per stage, we performed a comparison starting from nine dye-swaps and using a 22K bovine oligoarray. Statistical analysis revealed that the expression of 1,696 genes varied significantly at least once in the study. These genes fell into 19 clusters based on their expression profiles. Identification of biological functions with Ingenuity Pathway Analysis software revealed several similarities, in keeping with physiological stages described in mice. As in mice, expression of milk protein genes began at midpregnancy, and genes regulating lipid biosynthesis were induced at the onset of lactation. During the first half of pregnancy, the molecular signature of goat mammary tissue was characterized by the expression of genes associated with tissue remodeling and differentiation, while the second half was mainly characterized by the presence of messengers encoding genes involved in cell proliferation. A large number of immune-related genes were also induced, supporting recent speculation that mammary tissue has an original immune function, and the recruitment of migrating hematopoietic cells possibly involved in the branching morphogenesis of the mammary gland. These data hint that the induction of differentiation occurs early in pregnancy, very likely before P46. This period is therefore crucial for obtaining a healthy and productive mammary gland.

The olig family: phylogenetic analysis and early gene expression in Xenopus tropicalis.

The olig genes form a small subfamily of basic helix-loop-helix transcription factors. They were discovered in 2000 as genes required for oligodendrocyte lineage specification. Since then, olig genes have been identified in various vertebrate species and corresponding sequences accumulated within genomic databases. Until now, three groups of olig genes have been characterized. Our phylogenetic analysis demonstrates the existence of a fourth group, which we named olig4. Genes of the four olig groups are present in actinopterygians and amphibians, whereas mammals only possess olig1, 2, and 3. We also found one olig gene in hemichordates, urochordates, and cephalochordates. Our expression study during Xenopus tropicalis embryogenesis shows that the four olig genes have very distinct expression patterns. Olig1 is very faintly expressed before the tadpole stage, whereas olig2, 3, and 4 are expressed from the gastrula stage onward. The olig3 expression during neurulation suggests a role in early anteroposterior patterning of the brain. All these results indicate that olig genes are involved in several developmental processes during early development.

A cDNA macroarray resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk and beef) traits.

cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.

Human and mouse XAP-5 and XAP-5-like (X5L) genes: identification of an ancient functional retroposon differentially expressed in testis.

Although most retroposons that arose by reverse transcription of cellular mRNAs and by reintegration into the genome are nonfunctional, several examples exist in which the retroposon acquired a novel function and became fixed in the genome as a functional gene. We identified another such case: the ubiquitously expressed X-linked XAP-5 gene with unknown function gave rise to its retroposed counterpart, XAP-5-like (X5L), which has an intronless open reading frame and is autosomal in human. Phylogenetic analysis of the human and mouse XAP-5 and X5L genes shows that the retroposition most likely took place before the radiation of eutherian mammals. The XAP-5 and X5L genes are expressed in a wide range of tissues but are differentially expressed in testis. The ancient origin and broad expression of the X5L retroposon indicate that the XAP-5 and X5L genes may have assumed different functions in somatic cells. In addition to this, because of its autosomal location and its high level and particular pattern of expression in spermatogenic cells, the X5L expression in testis may compensate for the X-linked XAP-5 gene, which may be silenced during spermatogenesis.

Mutations in JAGGED1 gene are predominantly sporadic in Alagille syndrome.

BACKGROUNDS & AIMS: Mutations in the JAGGED1 gene are responsible for the Alagille syndrome, an autosomal dominant disorder characterized by neonatal jaundice, intrahepatic cholestasis, and developmental disorders affecting the liver, heart, vertebrae, eyes, and face. We screened a large group of patients for mutations in JAGGED1 and studied transmission of the mutations. METHODS: The coding sequence of the JAGGED1 gene was searched by single-strand conformation polymorphism and sequence analysis for mutations in 109 unrelated patients with the Alagille syndrome and their family if available. RESULTS: Sixty-nine patients (63%) had intragenic mutations, including 14 nonsense mutations, 31 frameshifts, 11 splice site mutations, and 13 missense mutations. We identified 59 different types of mutation of which 54 were previously undescribed; 8 were observed more than once. Mutations were de novo in 40 of 57 probands. CONCLUSIONS: Most of the observed mutations other than the missense mutations in JAGGED1 are expected to give rise to truncated and unanchored proteins. All mutations mapped to the extracellular domain of the protein, and there appeared to be regional hot spots, although no clustering was observed. Thus, the sequencing of 7 exons of JAGGED1 would detect 51% of the mutations. Transmission analysis showed a high frequency of sporadic cases (70%).

Transcription mapping in a medulloblastoma breakpoint interval and Smith-Magenis syndrome candidate region: identification of 53 transcriptional units and new candidate genes.

The chromosomal band 17p11.2 is associated with a number of neurological disorders and malignant diseases. This region is also characterized by the presence of complex repeat elements that are probably responsible for the frequent occurrence of interstitial deletions, duplications, and isochromosome formation. In the course of the molecular analysis of this interval, an integrated map with YACs, PACs, and cosmids covering approximately 6 Mb was established. Focusing on the 1.4-Mb interval containing the Smith-Magenis syndrome critical region and the breakpoint region for medulloblastomas, we constructed a detailed transcript map between the marker PS2 and the proximal CMT1A repeat. FISH analysis of the PACs allowed determination of the position of the transcripts with respect to the SMS critical region and the presumptive chromosomal breakpoint in medulloblastomas. One PAC (G21100) provided evidence for the presence of a novel complex repeat unit, indicating that there are at least three independent repeat elements within 2 Mb. Five genes were mapped to clone G21100 and are likely to form part of this novel complex sequence repeat. In summary, 53 new transcripts were isolated by using cDNA selection and exon trapping. This included 8 known but previously unmapped genes and 45 novel transcripts. The expression profile of 21 transcripts was determined by RT-PCR. Based on their homologies to known genes or proteins, some of the novel genes are considered candidate genes either for malignant diseases or for the Smith-Magenis syndrome.

Construction of an integrated physical and gene map of human chromosome 20p12 providing candidate genes for Alagille syndrome.

Physical mapping and localization of eSTS markers were used to generate an integrated physical and gene map covering a approximately 10-Mb region of human chromosome 20p12 containing the Alagille syndrome (AGS) locus. Seventy-four STSs, 28 of which were derived from cDNA sequences, mapped with an average resolution of 135 kb. The 28 eSTS markers define 20 genes. Six known genes, namely CHGB, BMP2, PLCB1, PLCB4, SNAP, and HJ1, were precisely mapped. Among the genes identified, one maps in the smallest region of overlap of the deletions associated with AGS and could therefore be regarded as a candidate gene for Alagille syndrome.

Extinction of peroxisomal functions in hepatoma cell-fibroblast hybrids.

Although peroxisomes are ubiquitous, differences in the number of organelles and in the expression of associated metabolic activities are observed, depending on the cell type. To investigate the control of peroxisomal activity in connection with cell differentiation, we constructed hybrids between two types of cells whose histogenetic origins dictate significant differences in peroxisomal activities: hepatoma cells and fibroblasts, with high and low expression, respectively, of peroxisomal functions. In these hybrids, extinction of the elevated activities that characterize liver cells is observed, in parallel with the well-documented extinction of differentiated functions. This suggests the existence in fibroblasts of a negative trans-acting regulation.

Construction of a 3.7-Mb physical map within human chromosome 20p12 ordering 18 markers in the Alagille syndrome locus.

Alagille syndrome (AGS, MIM 118450) is associated with human chromosome band 20p12. To study this region, we constructed a 3.7-Mb physical map using 36 YACs isolated from the CEPH YAC library with three sequence-tagged sites (STS): D20S503, D20S41, and D20S188. New STSs were obtained from 6 isolated YAC end-fragments. Eighteen markers were ordered on the contig as follows:20ptel-D20S177-D20S175-D20S509- D20S5/D20S503-D20S506-D20S162-D20S504- D20S505-D20S507-D20S188-(D20S6-D20S27- D20S189)-D20S186-D20S41-D20S61-D20S492- D20S508-20pcen. A restriction map with the enzymes AscI, MluI, NotI, SacII, and SfiI was generated, revealing seven putative CpG islands. We established a YAC contig that spans the AGS region and thus will be valuable for cloning candidate genes and searching for DNA polymorphisms segregating with this syndrome.

Segregation analysis of Alagille syndrome.

Alagille syndrome (AGS) is a well defined genetic disorder characterised by five major features. An autosomal dominant mode of transmission with reduced penetrance has been suggested by the analysis of a limited number of families. However there has been no statistical analysis. We report here the first segregation analysis of AGS, using 33 families collected through 43 probands. Segregation analysis of these families allowed us to conclude that AGS is transmitted as a dominant disorder with 94% penetrance and 15% of cases are sporadic. The expressivity of the phenotype was variable and 26 persons (15 parents and 11 sibs) were identified as presenting minor forms of the disease. These results are valuable for genetic counselling.