Highlighting new phylogenetic specificities of Crohn's disease microbiota.

BACKGROUND: Recent studies suggest that gastrointestinal (GI) microbes play a part in the pathogenesis of Crohn's disease (CD). METHODS: Fecal samples were collected from 16 healthy individuals and 16 CD patients (age- and sex-matched). The DNA extracted from these samples were subjected to two different methods of microbiome analysis. Specific bacterial groups were quantified by real-time polymerase chain reaction (PCR) methods using primers designed using a high-throughput in-house bioinformatics pipeline. The same DNA extracts were also used to produce fluorescently labeled cRNA amplicons to interrogate a custom-designed phylogenetic microarray for intestinal bacteria. RESULTS: Even though the intersubject variability was high, differences in the fecal microbiomes of healthy and CD patients were detected. Faecalibacterium prausnitzii and Escherichia coli were more represented in healthy and ileal CD patients, respectively. Additionally, probes specific for Ruminococcus bromii, Oscillibacter valericigenes, Bifidobacterium bifidum, and Eubacterium rectale produced stronger hybridization signals with the DNA samples from healthy subjects. Conversely, species overrepresented in CD patients were E. coli, Enterococcus faecium, and species from the Proteobacteria not normally found in the healthy human GI tract. Furthermore, we detected "healthy specific" molecular species or operational taxonomic units (OTUs) that are not closely related to any known species (Faecalibacterium, Subdoligranulum, and Oscillospora species), indicating that the phylogenetic dysbiosis is broader than at strain or species level. CONCLUSIONS: These two techniques of microbiome analysis provided a statistically robust new picture of the dysbiosis in fecal microbiota from ileal CD patients. Specifically, we identified a set of six species discriminant for CD, which provides a preliminary diagnostic tool.

The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age.

BACKGROUND: In humans, the intestinal microbiota plays an important role in the maintenance of host health by providing energy, nutrients, and immunological protection. Applying current molecular methods is necessary to surmount the limitations of classical culturing techniques in order to obtain an accurate description of the microbiota composition. RESULTS: Here we report on the comparative assessment of human fecal microbiota from three age-groups: infants, adults and the elderly. We demonstrate that the human intestinal microbiota undergoes maturation from birth to adulthood and is further altered with ageing. The counts of major bacterial groups Clostridium leptum, Clostridium coccoides, Bacteroidetes, Bifidobacterium, Lactobacillus and Escherichia coli were assessed by quantitative PCR (qPCR). By comparing species diversity profiles, we observed age-related changes in the human fecal microbiota. The microbiota of infants was generally characterized by low levels of total bacteria. C. leptum and C. coccoides species were highly represented in the microbiota of infants, while elderly subjects exhibited high levels of E. coli and Bacteroidetes. We observed that the ratio of Firmicutes to Bacteroidetes evolves during different life stages. For infants, adults and elderly individuals we measured ratios of 0.4, 10.9 and 0.6, respectively. CONCLUSION: In this work we have confirmed that qPCR is a powerful technique in studying the diverse and complex fecal microbiota. Our work demonstrates that the fecal microbiota composition evolves throughout life, from early childhood to old age.

Low counts of Faecalibacterium prausnitzii in colitis microbiota.

BACKGROUND: The intestinal microbiota is suspected to play a role in colitis and particularly in inflammatory bowel disease (IBD) pathogenesis. The aim was to compare the fecal microbiota composition of patients with colitis to that of healthy subjects (HS). METHODS: fecal samples from 22 active Crohn's disease (A-CD) patients, 10 CD patients in remission (R-CD), 13 active ulcerative colitis (A-UC) patients, 4 UC patients in remission (R-UC), 8 infectious colitis (IC) patients, and 27 HS were analyzed by quantitative real-time polymerase chain reaction (PCR) targeting the 16S rRNA gene. Bacterial counts were transformed to logarithms (Log(10) CFU) for statistical analysis. RESULTS: Bacteria of the phylum Firmicutes (Clostridium leptum and Clostridium coccoides groups) were less represented in A-IBD patients (9.7; P = 0.004) and IC (9.4; P = 0.02), compared to HS (10.8). Faecalibacterium prausnitzii species (a major representative of the C. leptum group) had lower counts in A-IBD and IC patients compared to HS (8.8 and 8.3 versus 10.4; P = 0.0004 and P = 0.003). The Firmicutes/Bacteroidetes ratio was lower in A-IBD (1.3; P = 0.0001) and IC patients (0.4; P = 0.002). Compared to HS, Bifidobacteria were less represented in A-IBD and IC (7.9 and 7.7 versus 9.2; P = 0.001 and P = 0.01). CONCLUSIONS: The fecal microbiota of patients with IBD differs from that of HS. The phylum Firmicutes and particularly the species F. prausnitzii, are underrepresented in A-IBD patients as well as in IC patients. These bacteria could be crucial to gut homeostasis since lower counts of F. prausnitzii are consistently associated with a reduced protection of the gut mucosa.

Quantitative effects of an intronic retroviral insertion on the transcription of the tyrosinase gene in recessive white chickens.

Recently, we reported the complete association of a retroviral insertion in intron 4 of the tyrosinase gene and the recessive white mutation (c) in chickens. The mutant allele carrying the retroviral insertion produced, in skin samples of 10-week-old chickens, aberrant tyrosinase transcripts that did not contain exon 5. In the present study, we performed serial molecular and statistical analyses on embryos and 10-week-old chickens to characterize the quantitative effect of the retroviral insertion on the expression pattern of tyrosinase in different tissues (skin and retina). By using quantitative real-time RT-PCR, we observed that the expression level of tyrosinase was significantly lower in recessive white chickens than in wild-type coloured chickens, but that this pattern was age- and tissue-dependent. The differential expression in skin was not significant in embryos, whereas it was highly significant in 10-week-old chickens. Furthermore, there was no difference in the expression of tyrosinase in the retinal pigment epithelium of animals with different genotypes; this corresponds to phenotypic data, which show pigmented eyes in both genotypes. These findings show that the retroviral insertion disturbs tyrosinase expression in the recessive white mutant chickens, and suggests that the regulation of tyrosinase expression in chickens differs between embryos and growing animals, as well as between skin and retina.

Differential activities of four Lactobacillus casei promoters during bacterial transit through the gastrointestinal tracts of human-microbiota-associated mice.

In a previous study using fusion of the deregulated lactose promoter lacTp* and reporter genes, we suggested that Lactobacillus casei could initiate de novo protein synthesis during intestinal transit. In order to confirm this finding and extend it to other promoters, we adopted a reverse transcriptase quantitative PCR (RT-QPCR) approach combined with a transcriptional fusion system consisting of luciferase genes under the control of four promoters (ccpA, dlt, ldh, and lacT*) from L. casei DN-114 001. Promoter expression was monitored during cell growth, and variable luciferase activities were detected. In 3-day cultures, all the genetically modified strains survived but without exhibiting luciferase activity. Luciferase mRNA levels determined by RT-QPCR analysis (RNA/CFU) were not significant. The cultures were administered to human-microbiota-associated mice, and the feces were collected 6 h later. L. casei promoters lacTp* and ldhp initiated mRNA synthesis during gastrointestinal transit. The promoters, ccpAp and dltp, exhibited no luciferase activity, nor was de novo-synthesized luciferase mRNA detected in the feces. L. casei seems to adapt its physiology to the gastrointestinal tract environment by modulating promoter activities. The approach (fecal transcriptional analysis) described herein may, moreover, be of value in studying gene expression of transiting bacteria in human fecal specimens.

A 11.7-kb deletion triggers intersexuality and polledness in goats.

Mammalian sex determination is governed by the presence of the sex determining region Y gene (SRY) on the Y chromosome. Familial cases of SRY-negative XX sex reversal are rare in humans, often hampering the discovery of new sex-determining genes. The mouse model is also insufficient to correctly apprehend the sex-determination cascade, as the human pathway is much more sensitive to gene dosage. Other species might therefore be considered in this respect. In goats, the polled intersex syndrome (PIS) mutation associates polledness and intersexuality. The sex reversal affects exclusively the XX individuals in a recessive manner, whereas the absence of horns is dominant in both sexes. The syndrome is caused by an autosomal gene located at chromosome band 1q43 (ref. 9), shown to be homologous to human chromosome band 3q23 (ref. 10). Through a positional cloning approach, we demonstrate that the mutation underlying PIS is the deletion of a critical 11.7-kb DNA element containing mainly repetitive sequences. This deletion affects the transcription of at least two genes: PISRT1, encoding a 1.5-kb mRNA devoid of open reading frame (ORF), and FOXL2, recently shown to be responsible for blepharophimosis ptosis epicanthus inversus syndrome (BPES) in humans. These two genes are located 20 and 200 kb telomeric from the deletion, respectively.

Fine mapping suggests that the goat Polled Intersex Syndrome and the human Blepharophimosis Ptosis Epicanthus Syndrome map to a 100-kb homologous region.

To clone the goat Polled Intersex Syndrome (PIS) gene(s), a chromosome walk was performed from six entry points at 1q43. This enabled 91 BACs to be recovered from a recently constructed goat BAC library. Six BAC contigs of goat chromosome 1q43 (ICC1-ICC6) were thus constructed covering altogether 4.5 Mb. A total of 37 microsatellite sequences were isolated from this 4.5-Mb region (16 in this study), of which 33 were genotyped and mapped. ICC3 (1500 kb) was shown by genetic analysis to encompass the PIS locus in a approximately 400-kb interval without recombinants detected in the resource families (293 informative meioses). A strong linkage disequilibrium was detected among unrelated animals with the two central markers of the region, suggesting a probable location for PIS in approximately 100 kb. High-resolution comparative mapping with human data shows that this DNA segment is the homolog of the human region associated with Blepharophimosis Ptosis Epicanthus inversus Syndrome (BPES) gene located in 3q23. This finding suggests that homologous gene(s) could be responsible for the pathologies observed in humans and goats.

High-resolution human/goat comparative map of the goat polled/intersex syndrome (PIS): the human homologue is contained in a human YAC from HSA3q23.

The genetic and cytogenetic map around the chromosome 1 region shown to be linked with polledness and intersexuality (PIS) in the domestic goat (Capra hircus) was refined. For this purpose, a goat BAC library was systematically screened with primers from human coding sequences, scraped chromosome 1 DNA, bovine microsatellites from the region, and BAC ends. All the BACs (n = 30) were mapped by fluorescence in situ hybridization (FISH) on goat chromosome 1q41-q45. The genetic mapping of 30 new goat polymorphic markers, isolated from these BACs, made it possible to reduce the PIS interval to a region of less than 1 cM on goat chromosome 1q43. The PIS locus is now located between the two genes ATP1B and COP, which both map to 3q23 in humans. Genetic, cytogenetic, and comparative data suggest that the PIS region is now probably circumscribed to an approximately 1-Mb DNA segment for which construction of a BAC contig is in progress. In addition, a human YAC contig encompassing the blepharophimosis-ptosis-epicanthus-inversus region was mapped by FISH to goat chromosome 1q43. This human disease, mapped to HSA 3q23 and affecting the development and maintenance of ovarian function, could be a potential candidate for goat PIS.

Analysis of genetic relationships between 10 cattle breeds with 17 microsatellites.

To guide genetic conservation programmes with objective criteria, general genetic variability has to be taken into account. This study was conducted to determine the genetic variation between 10 cattle breeds by using 17 microsatellite loci and 13 biochemical markers (11 blood groups, the transferrin and beta-casein loci). Microsatellite loci were amplified in 31-50 unrelated individuals from 10 cattle breeds: Charolais, Limousin, Breton Black Pied, Parthenais, Montbéliard, Vosgien, Maine-Anjou, Normande, Jersey and Holstein. Neighbor-joining trees were calculated from genetic distance estimates. The robustness of tree topology was obtained by bootstrap resampling of loci. A total of 210 alleles of the 17 microsatellites were detected in this study and average heterozygosities ranged from 0.53 in the Jersey breed to 0.66 in the Parthenais breed. In general, low bootstrap values were obtained: with the 17 microsatellites, the highest bootstrap values concerned the Holstein/Maine-Anjou grouping with an occurrence of 74%; with the biochemical markers, this node had an occurrence of 79% and the Charolais/Limousin grouping appeared with an occurrence of 74%; when microsatellites and biochemical polymorphism were analysed together, the occurrence of the Holstein/Maine-Anjou grouping was 90% and that of the Charolais/Limousin grouping was 42%. These results suggest that 30 microsatellites, a number currently considered as sufficient to distinguish closely related breeds is, in fact, probably insufficient.

A cytogenetically anchored genetic map of bovine chromosome 1 obtained by integrating flow-sorted chromosome-derived microsatellite markers into the international bovine map.

A genomic library was constructed from a peak of flow-sorted bovine chromosomes 1 + X after PCR amplification. Forty-three bovine chromosome 1 microsatellites were isolated, genetically mapped and integrated in the international genetic map. In addition, BAC clones from a goat BAC library were identified for five markers (DVEPC119, INRA011, BM4307, KAP8 and MAF64). These goat BACs could be mapped by FISH onto bovine chromosome 1 to bands 1q44-->q45, 1q25, 1q21, 1q12 and 1q14-->q21, respectively. This map reduces the average interval between consecutive markers on the international bovine genetic map from 5.5 cM to 2.5 cM, and provides a good starting point for positional cloning projects in cattle, sheep or goats.

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA.

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.

Complete nucleotide sequence of bovine alpha-lactalbumin gene: comparison with its rat counterpart.

The nucleotide sequence of the bovine alpha-lactalbumin gene, whose organization is very similar to that of its rat counterpart, was deduced from the analysis of 2 lambda clones isolated from a HindIII genomic bank. The 3090 sequenced nucleotides comprise 738 bp upstream from the transcription unit (approximately 2 kb) which contains 4 exons of 160, 159, 76 and 330 bp separated by 3 introns of 321, 473 and 504 bp. Comparison with the rat alpha-lactalbumin gene shows similar percentages of homology between the 4 cognate exons. Since only the first three exons are homologous to the corresponding exons of the lysozyme gene, it is suggested that the 4th exons of alpha-lactalbumin and lysozyme genes have different origins. The bovine alpha-lactalbumin mRNA is 725 nucleotides long, excluding the poly(A) tail. The reading frame and the flanking 5' and 3' untranslated regions contain 429, 27 and 269 nucleotides, respectively. The derived amino acid sequence differs at 10 positions from that determined directly on mature alpha-lactalbumin.

Ovine beta-lactoglobulin messenger RNA: nucleotide sequence and mRNA levels during functional differentiation of the mammary gland.

The nucleotide sequence of ovine beta-lactoglobulin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and primer extension products. Ovine beta-lactoglobulin mRNA consists of a 540 nucleotide coding region, flanked by 39 nucleotide 5' and 206 nucleotide 3' non-coding regions including a 20 nucleotide poly A tail. The deduced 180 amino acid sequence of pre-beta-lactoglobulin is in agreement with the previously published amino acid sequence of signal peptide and mature protein. Northern blot analysis of poly A+ RNAs from the lactating mammary glands of porcine, rabbit and rat species, allowed us to identify a homologous RNA to beta-lactoglobulin mRNA solely in the porcine species. We also detected a mRNA transcript of a size similar to that of beta-lactoglobulin mRNA in hepatic poly A+ RNA from female rat liver treated by estrogens. Furthermore, we have examined the levels of beta-lactoglobulin mRNA during the functional differentiation of the mammary gland and after hormonal stimulation. During the last third of pregnancy, the expression of beta-lactoglobulin gene is significantly more elevated than that of alpha s1- or beta-casein whose mRNA levels were found to change very slightly during this period. Both beta-lactoglobulin and casein mRNAs showed a rapid response and a wide range of change in response to cortisol treatment. However, there was a significant difference in the rate at which these processes occurred, suggesting that beta-lactoglobulin gene expression is regulated independently of the casein genes.