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International audience; The availability of the sequences for whole genomes is changing our understanding of cell biology. Functional genomics refers to the comprehensive analysis, at the protein level (proteome) and at the mRNA level (transcriptome) of all events associated with the expression of whole sets of genes. New methods have been developed for transcriptome analysis. Serial Analysis of Gene Expression (SAGE) is based on the massive sequential analysis of short cDNA sequence tags. Each tag is derived from a defined position within a transcript. Its size (14 bp) is sufficient to identify the corresponding gene and the number of times each tag is observed provides an accurate measurement of its expression level. Since tag populations can be widely amplified without altering their relative proportions, SAGE may be performed with minute amounts of biological extract. Dealing with the mass of data generated by SAGE necessitates computer analysis. A software is required to automatically detect and count tags from sequence files. Criterias allowing to assess the quality of experimental data can be included at this stage. To identify the corresponding genes, a database is created registering all virtual tags susceptible to be observed, based on the present status of the genome knowledge. By using currently available database functions, it is easy to match experimental and virtual tags, thus generating a new database registering identified tags, together with their expression levels. As an open system, SAGE is able to reveal new, yet unknown, transcripts. Their identification will become increasingly easier with the progress of genome annotation. However, their direct characterization can be attempted, since tag information may be sufficient to design primers allowing to extend unknown sequences. A major advantage of SAGE is that, by measuring expression levels without reference to an arbitrary standard, data are definitively acquired and cumulative. All publicly available data can thus be stored in a unique database, facilitating whole-genome analysis of differential expression between cell types, normal and diseased samples, or samples with and without drug treatment. SAGE data are readily amenable to statistical comparisons, allowing to determine the level of confidence of the observed variations. A major limitation of SAGE is that, because each analysis is obligatory performed on the whole set of expressed genes, it can hardly be performed on multiple samples, for example in kinetics studies or to compare the effects of large numbers of drugs. To overcome this limitation, high-throughput detection of a subset of mRNAs is more rapidly performed by parallel hybridization of mRNAs on arrays of nucleic acids immobilized on solid supports. From this point of view, a SAGE platform is a powerful instrument for selecting the most informative subset of genes, assembling them to design microarrays dedicated to a specific problem and calibrating measurement by comparison with a standard cell model for which SAGE data are available. This approach is an attractive alternative to strategies based exclusively on pangenomic arrays. A very large amount of SAGE data are already available and the problem is now to extract their biological meaning. Knowledge on metabolic pathways is already organized so that its successful integration in a SAGE platform can be undertaken. For other cell components and pathways, the problem lies on the lack of controlled vocabulary to describe gene activities, starting form a clear definition of the concept of biological function itself. Progress in gene and cell ontology is expected to facilitate computer-based extraction of biological knowledge from existing and forthcoming SAGE data.

African trypanosomiases are parasitic diseases transmitted by tse-tse flies, considered as the main sanitary obstacle to animal production development in sub-Saharan Africa. However, if trypanosomiases have dramatic consequences on zebu (Bos indicus) populations, they have a weaker impact on the western African taurine (Bos taurus), which is known to be naturally tolerant to trypanosome infection. Mechanisms governing this trypanotolerant trait are still poorly understood, but today, recent postgenomic biotechnologies, such as the SAGE technique (serial analysis of gene expression) allow us to explore the full transcriptome. Twelve SAGE libraries were constructed from two trypanotolerant animals (N'Dama and Baoulé) and one susceptible species of cattle (the Sudanese zebu) during an experimental Trypanosoma congolense infection; 43,458 different tags were obtained at several particular points during the infection (before infection, at the maximum of parasitemia, the maximum of anemia, and at the end of the experiment after value normalization). Bioinformatics analyses highlighted some interesting gene variations with respect to the trypanotolerance status of the animal.

Background: The kidney development involves a wide variety of developmental processes requiring a lot of genes expressed in a sequential manner. The aim of the present study is to identify new genes involved in these processes. Methods: To obtain a view of the mouse embryonic kidney transcriptome we used the SADE method, which allows large-scale quantitative gene expression measurements. Results: 7,689 tags were sequenced from our library. Among the 4,507 unique transcripts yielded, 64% correspond to known genes, 22% ESTs, 12% unidentified genes. 472 genes were differentially expressed as compared to published adult kidney library. Among these, we identified several candidate genes and focused on a particular one: thymosin β4 (Tβ4), an actin-sequestering protein more highly expressed in fetal kidney. First we studied the in vivo expression patterns of Tβ4 transcript during kidney development. Tβ4 increases throughout the kidney development and remains high during active nephrogenesis. Moreover, the spatial distribution of Tβ4 mRNA was analysed and reveals that during active nephrogenesis (i.e., 18 dpc) Tβ4 is localised in differentiating glomeruli. In adult kidney, Tβ4 remains expressed in podocytes and collecting ducts. Conclusion: Our results provide the first demonstration of Tβ4 production in vivo by embryonic kidney and further show that Tβ4 is implicated in kidney organogenesis.

Because of its short life span in blood, its low RNA content and its highly condensed nucleus, the granulocyte was initially considered as a terminally differentiated cell unable to express novel genes. However, mature granulocytes still contain a variety of mRNAs and may respond to external stimuli by rapid and complex changes in gene expression. The present work was undertaken to provide a wider view of the gene expression profile in unstimulated circulating PMNs. We used serial analysis of gene expression (SAGE) adapted to downsized extracts (SADE) to cope with their small mRNA content. As cell samples of the highest degree of purity were crucial for this project, we adapted a magnetic cell sorting method to reach the required high levels of purity (99.55 +/- 0.19%) together with low activation rates (1.37 +/- 0.28). We analyzed 20,787 tags identifying 8547 different transcripts, of which 47.8% were unknown, 34.6% were transcripts of known genes, and 13.8% matched with expressed sequence tags (EST). Highly expressed genes were those involved in cell mobility, diapedesis, cell signaling and destruction of micro-organisms. In addition, this method led to the identification of genes which had not previously been reported in granulocytes. These results could provide new molecular markers and a reliable reference for the investigation of pathologies involving alteration of the granulocyte gene expression profile.

The low-affinity Fcγ receptor IIIb (FcγRIIIb and CD16b) is constituted by a unique FCGR3B polypeptide chain that comprises two extracellular immunoglobulin-like domains, and is expressed as a glycosylphosphatidyl inositol-anchored receptor on the neutrophils. The FCGR3B chain bears allotypes that define the human neutrophil antigen-1 (HNA-1 and NA) system involved in major post-transfusional reactions. FCGR3B is highly homologous to FCGR3A, which is expressed as a transmembrane receptor on natural killer cells and monocytes/macrophages. Its transcription products were not yet fully characterized. In the present work, we sequenced FCGR3B cDNAs with complete 3′ untranslated region from purified granulocytes of HNA-1b/HNA-1b (NA2/NA2) genotyped donors. We characterized two FCGR3B cDNAs of different lengths corresponding to two polyadenylation sites. This result was corroborated by data raised by serial analysis of gene expression (SAGE). FCGR3B allele polymorphisms, from this article [FCGR3B*02 (HNA-1b, NA2)] and from the literature, are described for the first time according to the IMGT standardized nomenclature and to the IMGT unique numbering for C-LIKE-DOMAIN (http://www.imgt.cines.fr). These rules, described in the IMGT Scientific chart, are based on the IMGT-ONTOLOGY concepts. IMGT allele alignments and IMGT Collier de Perles graphical two-dimensional representations are provided for the two Ig-like domains (or C-LIKE-DOMAINs) [D1] and [D2] of FCGR3B*02. The standardized description of FCGR3B allele polymorphisms was approved by the IMGT Nomenclature Committee (IMGT-NC) and is freely available in IMGT repertoire at IMGT, http://www.imgt.cines.fr.

Vitamin D and retinoids cooperate to inhibit the proliferation and induce the differentiation of human myelomonocytic U937 leukemia cells. In the present work, we investigated the role of TGF-β as an endogenous mediator of this process. We found that the TGF-β1 precursor began to accumulate in cell culture supernatants soon after the addition of 1α,25 dihydroxyvitamin D3 (VD) and retinoids. We used neutralizing antibodies (AbTGF-β) and antisense oligonucleotide (AS Oligo) to inhibit its possible effects. Our data demonstrated that AbTGF-β partially inhibit the expression of the differentiated phenotype, as assessed by measurement of phagocytic activity, response to the chemotactic peptide fMLP, and lysozyme secretion. AS Oligo was also inhibitory, and the effects of AS Oligo and AbTGF-β were cumulative. Cell growth inhibition induced by VD and retinoids was completely reversed, and differentiation was reduced by about 75% when both inhibitors were associated. Time course experiments based on the delayed addition of AbTGF-β and AS Oligo showed that TGF-β1 was required for cell differentiation 24 h after the addition of inducers. Studies on TGF-β receptors revealed that, while the expression of type II receptor was stable, the level of type I TGF-β receptor mRNA and the expression of the protein began to decline early during the differentiation process. As a whole, these results support the notion that an autocrine TGF-β pathway, activated by VD and retinoids in U937 cells, is involved in the early steps of the process leading to cell growth arrest and differentiation. J Cell Physiol 178:109–119, 1999. © 1999 Wiley-Liss, Inc.

Retinoids and vitamin D (VD) cooperate to induce the differentiation and inhibit the proliferation of human myelomonocytic leukemia cells. Two classes of retinoids receptors, the RARs and RXRs, respectively, can mediate these effects. RXR forms heterodimers with a variety of nuclear receptors, including RAR and the VD receptor. We have previously found that VD treatment increases RXR alpha levels in myelomonocytic leukemia cells. By immunoanalysis, we observed in the present work that the RAR alpha protein is expressed in proliferating U937, HL-60 and THP-1 human leukemia cells and that VD treatment induces alterations of its electrophoretic pattern, although with large differences between cell lines. In the three cell lines, 9-cis RA, an agonist of both RARs and RXRs, cooperated with VD more efficiently than all-trans RA and RAR-specific synthetic ligands, thus suggesting an involvement of both RAR and RXR pathways in cell differentiation. Using U937 cells as a model, we delineated the relative contributions of RAR and RXR by assessing the effects of receptor-selective synthetic retinoids. The synergy between VD and all-trans RA or RAR-specific agonists (TTNPB and Ro 40-6055) was abrogated by a RAR alpha-specific antagonist (Ro 41-5253), confirming an involvement of RAR alpha. However, the cooperation between VD and 9-cis RA, although reduced, was not suppressed by the antagonist, suggesting also an involvement of the RXR pathway. The role of RXR as a ligand-activated receptor was confirmed using RXR-specific agonists (CD2608 and LGD1069), which also proved able to cooperate with VD. Finally, while each synthetic agonist alone was significantly less potent than 9-cis RA, combinations of the RAR and RXR selective agonists TTNPB and LGD1069 appeared to be as effective as the pan agonist 9-cis-RA. These results confirm that various retinoids can cooperate with VD and demonstrate that, at a whole cell level, optimal effects require the activation of both RAR and RXR receptors.

β-Lactoglobulin (βLG) was submitted to various thermal treatments (75 and 90 °C), and its conformational modifications were assessed by competitive radioimmunoassay (RIA) with rabbit antisera against native βLG (βLG NAT ) or chemically modified βLG obtained by performic oxidation (βLG OX ). Heat-processed βLG reacted with both antisera. Molecule alteration was accompanied by an increased binding of polyclonal antibodies. Whatever the serum used, the RIA test revealed a thermal dependence that was influenced by pH (6.5 and 7.5). However, anti-βLG OX antiserum was shown to be the most useful tool for monitoring slight denaturation structural changes after heating. The test accurately detected local modifications of βLG epitopes that occurred after thermal processing of an industrial βLG isolate.

International audience; no abstract

Among the nuclear hormone receptors, the retinoid X receptors (RXRs) play a central role through their ability to heterodimerize with other members of this family of transcription factors, including retinoic acid (RA) and vitamin D (VD3) receptors. We have previously found that all-trans retinoic acid and 1 alpha, 25-dihydroxyvitamin D3 cooperate to induce monocytic differentiation of U937 human leukemic cells. Here the expression of RXR alpha protein in myelomonocytic cells was studied by immunodetection using polyclonal antibodies. RXR alpha was detected upon exposure of cells to VD3 and higher levels were found in cells treated by combinations of RA and VD3 under conditions where both agents synergized for inducing monocytic properties.

The lectin jacalin is mitogenic for CD4 expressing T lymphocytes, interacts with the CD4 molecule, and inhibits HIV infection of CD4+ cells. In the present study the effect of jacalin was tested on cells from the monocyte/macrophage lineage that also express the CD4 molecule. We used CD4+ promyelomonocytic U937 cells differentiated towards the monocytic/macrophage lineage with either a mixture of two physiological agents, retinoic acid (RA) and 1α,25-dihydroxyvitamin D3 (VD), or the exogenous drug phorbol myristate acetate (PMA). The cells resulting from these treatments differed in term of CD4 expression. We focused our attention on interleukin-6 (IL-6) production, which implies an activation of the cells differentiated along both pathways. In CD4+ RA/VD-treated cells, jacalin induced a 10-fold higher IL-6 secretion than did lipopolysaccharide (LPS). This jacalin-induced IL-6 production was inhibited by agents interacting with CD4 (anti-CD4 mAbs and HIV recombinant gp120) or by recombinant soluble CD4. In contrast, the CD4- PMA-differentiated U937 cells did not secrete any IL-6 upon jacalin treatment, while they demonstrated a response to LPS similar to that of the RA/VD-differentiated cells. Together with the fact that jacalin interacts with CD4, these results provide evidence of the involvement of a CD4 dependent pathway in IL-6 production. J. Leukoc. Biol. 55: 214–220; 1994.

We report the effect of CBS-211A, a synthetic retinoid analog, designed for topical eye administration, on the growth and differentiation of myelomonocytic cells. This compound was assayed alone or in combination with 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3), since we previously evidenced a synergism of retinoic acid (RA) and 1 alpha,25(OH)2D3 in the induction of U937 cell differentiation. Unlike RA, CBS-211A neither affected the growth of myelomonocytic cells nor differentiated them. Nevertheless, when it was associated with 1 alpha,25(OH)2D3, CBS-211A strongly potentiated the 1 alpha,25(OH)2D3-induced inhibition of U937 cell proliferation and caused a dramatic increase in their differentiation toward monocytes/macrophages. The co-inducing effect of CBS-211A was restricted to U937 cells. Our data suggest that CBS-211A may have therapeutic implications in the treatment of certain kinds of myelomonocytic leukemia. CBS-211A also provides an interesting tool to understand the mechanisms involved in the differentiation of myelomonocytic cells.

RA induces the differentiation of myeloid leukemic cells both from patients and cell lines . The fact that differentiated cells loose their pro-liferative capacity provides an efficient approach for the treatment of acute promyelocytic leukemia by RA. Moreover a therapeutic effect extended to other fonns of leukemia is expected by using an appropriate synergistic combination between RA and other cell differentiating agents such as the biologically active form of vitamin D, la,25-dihy-droxyvitamin D

Program available at http://www.lirmm.fr/~rivals/GENOMICS; International audience; Analysis of several million expressed gene signatures (tags) revealed an increasing number of different sequences, largely exceeding that of annotated genes in mammalian genomes. Serial analysis of gene expression (SAGE) can reveal new Poly(A) RNAs transcribed from previously unrecognized chromosomal regions. However, conventional SAGE tags are too short to identify unambiguously unique sites in large genomes. Here, we design a novel strategy with tags anchored on two different restrictions sites of cDNAs. New transcripts are then tentatively defined by the two SAGE tags in tandem and by the spanning sequence read on the genome between these tagged sites. Having developed a new algorithm to locate these tag-delimited genomic sequences (TDGS), we first validated its capacity to recognize known genes and its ability to reveal new transcripts with two SAGE libraries built in parallel from a single RNA sample. Our algorithm proves fast enough to experiment this strategy at a large scale. We then collected and processed the complete sets of human SAGE tags to predict yet unknown transcripts. A cross-validation with tiling arrays data shows that 47% of these TDGS overlap transcriptional active regions. Our method provides a new and complementary approach for complex transcriptome annotation.

Disease relapse sometimes occurs after acute promyelocytic leukemia (APL) therapy with all-trans retinoic acid (ATRA). Among the diagnostic parameters predicting relapse, heterogeneity in the in vitro differentiation rate of blasts is an independent factor. To identify biologic networks involved in resistance, we conducted pharmacogenomic studies in APL blasts displaying distinct ATRA sensitivities. Although the expression profiles of genes invested in differentiation were similarly modulated in low- and high-sensitive blasts, low-sensitive cells showed higher levels of transcription of ATRA-target genes, transcriptional regulators, chromatin remodelers, and transcription factors. In opposition, only high-sensitive blasts expressed the CYP26A1 gene, encoding the p450 cytochrome which is known to be involved in retinoic acid catabolism. In NB4 cells, ATRA treatment activates a novel signaling pathway, whereby interleukin-8 stimulates the expression of the homeobox transcription factor HOXA10v2, an effective enhancer of CYP26A1 transcription. These data were corroborated in primary APL cells, as maturation levels correlated with CYP26A1 expression. Treatment with a retinoic acid metabolism blocking agent (RAMBA) results in high-nucleoplasmic concentrations of retinoid and growth of NB4-resistant subclones. Hence, for APL blasts associated with poor prognosis, the low CYP26A1 expression may explain high risk of resistance installation, by increased retinoid pressure. Pharmacogenomic profiles of genes involved in retinoid acid metabolism may help to optimize anticancer therapies, including retinoids.

Serial Analysis of Gene Expression (SAGE) is a method designed to measure the abundance of a large number of mRNAs in tissues and cells to characterize their transcriptome. By the end of the twentieth century, when determining the entire genomic sequence of higher organisms began to be perceived as a realistic and attainable goal, the interest for genome-wide analysis of gene expression rapidly increased. One of the first strategies was to generate expressed sequence tags (ESTs), that is, partial sequences of cDNA clones randomly selected in libraries prepared from various tissue samples. The EST approach was highly valuable for gene discovery. Moreover, as long as the abundance of a given mRNA could be correlated with the frequency of its EST, it might also provide quantitative information. However, this approach evaluated only a limited number of genes per library. Obviously, getting larger quantitative expression profiles required to increase the rate of acquisition of experimental data. SAGE rests on the sequencing of short diagnostic tags, and as such increased by more than 1 order of magnitude the number of transcripts analyzed by current sequencing methods. Sets of 100 000 SAGE tags are now routinely sequenced, making possible the quantification of low copy number mRNAs. Keywords: Expressed Sequence Tag (EST); Microarray; Polymerase Chain Reaction; Single Nucleotide Polymorphism; Transcriptome; Type IIS Restriction Endonuclease

In central and sub-Saharan Africa, trypanosomosis is a tsetse fly-transmitted disease, which is considered as the most important impediment to livestock production in the region. However, several indigenous West African taurine breeds (Bos taurus) present remarkable tolerance to the infection. This genetic capability, named trypanotolerance, results from numerous biological mechanisms most probably under multigenic dependences, among which are control of the trypanosome infection by limitation of parasitemia and control of severe anemia due to the pathogenic effects. Today, some postgenomic biotechnologies, such as transcriptome analyses, allow characterization of the full expressed genes involved in the majority of animal diseases under genetic control. One of them is serial analysis of gene expression (SAGE) technology, which consists of the construction of mRNA transcript libraries for qualitative and quantitative analysis of the entire genes expressed or inactivated at a particular step of cellular activation. We developed four different mRNA transcript libraries from white blood cells on a N'Dama trypanotolerant animal during an experimental Trypanosoma congolense (T. congolense) infection: one before experimental infection (ND0), one at the parasitemia peak (NDm), one at the minimal packed cell volume (NDa), and the last one at the end of the experiment after normalization (NDf). Bioinformatic comparisons in bovine genomic databases allowed us to obtain more than 75,000 sequences, among which are several known genes, some others are already described as expressed sequence tags (ESTs), and the last are completely new, but probably functional in trypanotolerance. The knowledge of all identified named or unnamed genes involved in trypanotolerance characteristics will allow us to use them in a field marker-assisted selections strategy and in microarrays prediction sets for bovine trypanotolerance.

Post genomic biotechnologies, such as transcriptome analysis, are now efficient enough to characterize the full complement of genes involved in the expression of specific biological functions. One of them is the Serial Analysis of Gene Expression (SAGE) technique. SAGE involves the construction of transcript libraries for a quantitative analysis of the entire set of genes expressed or inactivated at particular stages of cellular activation. Bioinformatic comparisons in hosts and pathogens genomic databases allow the identification of several up- and down-regulated genes, ESTs and unknown transcripts directly involved in the host-pathogen immunological interaction mechanisms. Based on the first results obtained during an experimental Trypanosoma congolense infection in trypanotolerant cattle, the efficiency and limits of such a technique, from the data acquisition level to the data analysis level, is discussed in this analysis.

We studied the gene expression profile of human purified reticulocytes to provide a transcriptional basis for the study of erythroid biology, differentiation and hematologic disorders.We screened highly purified blood reticulocytes from ten healthy adult volunteers. We chose a modified protocol of serial analysis of gene expression (SAGE), the serial analysis of downsized extracts (SADE).Data analysis revealed that 64% of gene signatures (tags) matched with known genes; mainly hemoglobin. In addition to the abundant globin mRNA, SAGE analysis identified previously described genes and new transcripts. In reticulocytes, which are poor in mRNA, we also identified 9% of EST and 27% of tags that did not match with any known genes. Mining our data, 70% of the unknown tags and 39% of tags identifying EST were found to be specific to the reticulocyte. We demonstrated the presence of a mRNA that matched with the reverse sequence of the hemoglobin b (HBB) transcript.This is the first description of an antisense transcript of the human HBB gene suggesting regulation by way of sense-antisense pairing. The well-characterized genes found in the SAGE library were genes specific to the blood cell lineage, housekeeping genes and, interestingly, genes not previously described in the reticulocyte. Furthermore the study provides markers of the erythroid lineage regulated during the differentiation process as observed in in vitro experiments.

As a growing number of complementary transcripts, susceptible to exert various regulatory functions, are being found in eukaryotes, high throughput analytical methods are needed to investigate their expression in multiple biological samples. Serial Analysis of Gene Expression (SAGE), based on the enumeration of directionally reliable short cDNA sequences (tags), is capable of revealing antisense transcripts. We initially detected them by observing tags that mapped on to the reverse complement of known mRNAs. The presence of such tags in individual SAGE libraries suggested that SAGE datasets contain latent information on antisense transcripts. We raised a collection of virtual tags for mining these data. Tag pairs were assembled by searching for complementarities between 24-nt long sequences centered on the potential SAGE-anchoring sites of well-annotated human expressed sequences. An analysis of their presence in a large collection of published SAGE libraries revealed transcripts expressed at high levels from both strands of two adjacent, oppositely oriented, transcription units. In other cases, the respective transcripts of such cis-oriented genes displayed a mutually exclusive expression pattern or were co-expressed in a small number of libraries. Other tag pairs revealed overlapping transcripts of trans-encoded unique genes. Finally, we isolated a group of tags shared by multiple transcripts. Most of them mapped on to retroelements, essentially represented in humans by Alu sequences inserted in opposite orientations in the 3′UTR of otherwise different mRNAs. Registering these tags in separate files makes possible computational searches focused on unique sense–antisense pairs. The method developed in the present work shows that SAGE datasets constitute a major resource of rapidly investigating with high sensitivity the expression of antisense transcripts, so that a single tag may be detected in one library when screening a large number of biological samples.

Large-scale identification of genes expressed in roots of the model plant Arabidopsis was performed by serial analysis of gene expression (SAGE), on a total of 144,083 sequenced tags, representing at least 15,964 different mRNAs. For tag to gene assignment, we developed a computational approach based on 26,620 genes annotated from the complete sequence of the genome. The procedure selected warrants the identification of the genes corresponding to the majority of the tags found experimentally, with a high level of reliability, and provides a reference database for SAGE studies in Arabidopsis. This new resource allowed us to characterize the expression of more than 3,000 genes, for which there is no expressed sequence tag (EST) or cDNA in the databases. Moreover, 85% of the tags were specific for one gene. To illustrate this advantage of SAGE for functional genomics, we show that our data allow an unambiguous analysis of most of the individual genes belonging to 12 different ion transporter multigene families. These results indicate that, compared with EST-based tag to gene assignment, the use of the annotated genome sequence greatly improves gene identification in SAGE studies. However, more than 6,000 different tags remained with no gene match, suggesting that a significant proportion of transcripts present in the roots originate from yet unknown or wrongly annotated genes. The root transcriptome characterized in this study markedly differs from those obtained in other organs, and provides a unique resource for investigating the functional specificities of the root system. As an example of the use of SAGE for transcript profiling in Arabidopsis, we report here the identification of 270 genes differentially expressed between roots of plants grown either with NO3 - or NH4NO3 as N source.

International audience

In Africa, trypanosomosis is a tsetse-transmitted disease which represents the most important constraint to livestock production. Several indigenous West African taurine (Bos taurus) breeds, such as the Longhorn (N'Dama) cattle are well known to control trypanosome infections. This genetic ability named "trypanotolerance" results from various biological mechanisms under multigenic control. The methodologies used so far have not succeeded in identifying the complete pool of genes involved in trypanotolerance. New post genomic biotechnologies such as transcriptome analyses are efficient in characterising the pool of genes involved in the expression of specific biological functions. We used the serial analysis of gene expression (SAGE) technique to construct, from Peripheral Blood Mononuclear Cells of an N'Dama cow, 2 total mRNA transcript libraries, at day 0 of a Trypanosoma congolense experimental infection and at day 10 post-infection, corresponding to the peak of parasitaemia. Bioinformatic comparisons in the bovine genomic databases allowed the identification of 187 up- and down- regulated genes, EST and unknown functional genes. Identification of the genes involved in trypanotolerance will allow to set up specific microarray sets for further metabolic and pharmacological studies and to design field marker-assisted selection by introgression programmes.

Highly sensitive and specific diagnostic tools are essential for monitoring the health status of farmed species. After the development of genomic probe diagnostic systems in the 1990s, followed by PCR-based systems, a miniarray method has been developed allowing one-step multiple detection. The miniarray method was developed to enable the accessibility of powerful array technology. To use this system, hybridisation and washing process were modified, resulting into a significant increase in the test's rapidity and sensitivity. With miniarray technology, hybridisation time is reduced to 20 min, whereas other methods require a longer hybridisation time. Hybridisation of the PCR product on a nylon membrane and revelation of the hybrids by an antibody increase considerably the ability of pathogen's detection. A first application is developed for the diagnosis of two specific viruses which are, by their geographical range and their impact on the production, very important in shrimp pathology, namely, the White Spot Syndrome Virus (WSSV) and the Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV).

The human leukemia cell line U937 is a well-established model for studying monocytic cell differentiation. We used a modified protocol (SADE) of serial analysis of gene expression (SAGE) and developed a SADE linker-anchored PCR assay to investigate the pattern of expression of known genes and to identify new transcripts in proliferating cells and during cell growth arrest and differentiation. We implemented new informatic tools to compare expression profiles before and after exposure of cells to differentiation inducers. From the analysis of 47,388 tags, we identified 13,806 distinct transcripts, 265 of which showed significant variations (P

Using a differential display method to identify differentiation-related genes in human myelomonocytic U937 cells, we cloned the cDNA of a gene identical to Drg1 and homologous to other recently discovered genes, respectively human RTP and Cap43 and mouse Ndr1 and TDD5 genes. Their open reading frames encode proteins highly conserved between mouse and man but which do not share homology with other know proteins. Conditions in which mRNAs are up-regulated suggest a role for the protein in cell growth arrest and terminal differentiation. We raised antibodies against a synthetic peptide reproducing a characteristic sequence of the putative polypeptide chain. These antibodies revealed a protein with the expected 43 kDa molecular mass, up-regulated by phorbol ester, retinoids and 1,25-(OH)2 vitamin D3 in U937 cells. It was increased in mammary carcinoma MCF-7 cells treated by retinoids and by the anti-estrogen ICI 182,780 but not by 4-hydroxytamoxifen. The mouse Drg1 homologous protein was up-regulated by retinoic acid in C2 myogenic cells. The diversity of situations in which expression of RTP/Drg1/Ndr1 has now been observed shows that it is widely distributed and up-regulated by various agents. Here we show that ligands of nuclear transcription factors involved in cell differentiation are among the inducers of this novel protein.

1 alpha, 25-Dihydroxyvitamin D3 (VD) is a potent inducer of monocytic differentiation of both normal and leukemic cells. Its effects are mediated by its nuclear receptor (VDR). Efficient gene activation requires the heterodimerization of VDR with Retinoid X Receptors (RXR). In the present study using specific antibodies, we analyzed the expression of the RXR alpha protein in blood mononuclear cells from acute myeloid patients (AML) (10 cases) and from myelomonocytic cell lines arrested at different stages of differentiation. We observed that the RXR alpha expression increased during myelomonocytic differentiation, since the highest levels were found in AML samples and in myelomonocytic cell lines having the highest amounts of monocytic precursors. We also demonstrated that fresh leukemic cells, whatever their stage of differentiation, as well as myelomonocytic cell lines, respond to VD by an increase in RXR alpha levels. Combinations of all-trans retinoic acid (RA) and VD, in some cases, increased this effect. This response suggests the involvement of RXR alpha in monocytic differentiation upon VD treatment.

The individual and combined effects of heat shock, all- trans retinoic acid and 1,25-dihydroxyvitamin D 3 on inhibition of cell growth and initiation of differentiation were investigated on U937 human leukemia cells. Incubation of U937 cells at 43°C for 1 h did not affect cell viability but induced a reduction of cell growth and the emergence of a differentiated phenotype, characterized by the acquisition of chemiluminescent responses to various oxidative burst inducers and by the capacity to produce IL-6 in response to bacterial lipopolysaccharide. Heat shock alone, therefore, appears to be an efficient inducer of cell differentiation. In addition, heat shock primed the cells to respond more efficiently to the action of retinoic acid and vitamin D, and amplified the phenotypic changes initiated by pretreatment of U937 cells with these agents.

The Brucella ovis dnaK gene, homolog to the eukaryotic hsp70 genes, was cloned by using a Drosophila melanogaster probe. Comparison of B. ovis and Escherichia coli sequences revealed a similar organization for the dnaK and dnaJ genes and putative regulatory signals. In E. coli transfected with the cloned fragment, B. ovis hsp70 was expressed at 30 and 50 degrees C apparently under the control of its own promoter. The recombinant protein and a B. ovis native protein displaying the same molecular weight were both recognized by anti-E. coli DnaK serum. Native B. ovis protein was also recognized by sera of sheep either infected or vaccinated with an attenuated Brucella strain, suggesting that Brucella hsp70 could be up-regulated during host colonization. A thermosensitive E. coli dnaK mutant transfected with the cloned fragment recovered colony-forming ability at 42 degrees C, showing that the B. ovis DnaK protein could behave as a functional heat shock protein in E. coli.

The human-derived leukemia cell lines HL-60 and U937 are known to differentiate into more mature phagocytic cells in the presence of retinoic acid or 1,25-dihydroxyvitamin D3. We studied the effects of combinations of these two agents on cell growth and differentiation. These treatments were found to increase inhibition of cell proliferation. A dramatic enhancement of functional properties was observed in U937, but not HL-60 cells exposed to combinations of the two inducers. We investigated the conditions required to obtain the highest synergistic effects on the differentiation of U937 cells. These effects were found to be highly dose-dependent. We found that synergism required the simultaneous presence of both inducers and did not occur upon sequential exposure to each agent used separately.

We have studied the expression of vimentin in the human histiocytic lymphoma cell line U937, induced to differentiate along the monocyte/macrophage pathway. Normal monocytes possess a network of vimentin intermediate filaments (IFs) at all stages of maturation. The undifferentiated U937 leukemia cells contain very low amounts of vimentin, but express a conspicuous IF network when exposed to phorbol myristate acetate. In parallel, they acquire functional properties typical of cells of the monocyte lineage. These concomitant variations suggest that vimentin IFs could play a role in the process of differentiation. However, we observed that all-trans-retinoic acid and 1,25-dihydroxyvitarnin D3, confer monocyte-like properties upon U937 cells without inducing vimentin expression. We obtained increased phenotypic changes, yet in the absence of a vimentin network, by combining the effects of both inducers. These results show that vimentin expression is not crucial for the acquisition of some of the functions characteristic of the monocyte/macrophage lineage.

Large scale analyses of transcriptome improve comprehension of complex processes such as differentiation or cell proliferation. SAGE libraries construction of the AML model U937 allowed the identification of new markers of myelomonocytic differentiation induced by retinoids and vitamin D3 (VD3) (Piquemal et al., 2002). Those molecules act through ligand-dependent transcription factors of the nuclear receptors family: RAR, RXR and VDR. Among differentially expressed members of transcriptional complexes, the most relevant was the co-regulator NCoA4 (Nuclear receptor Coactivator 4). This protein that modulates interactions between transcription factors, RNA polymerase II and chromatin remodeling factors, was initially described as a co-activator of the androgen receptor (AR). Its activity has been extended to receptors of estrogens (ER), peroxisome-proliferating activators (PPAR), retinoid X (RXR) and recently to VD3 receptor (VDR). Using real-time semi-quantitative PCR, we found that NCoA4 is specifically expressed during the monocytic differentiation of U937 but not during the granulocytic differentiation of NB4 cell lines. These results were confirmed by analysis on normal and in vitro-differentiated leukemia primary cells. Moreover, its early induction, within 6 hours of retinoids and VD3 treatment on U937 cells, suggests that its expression may be controlled by one or several nuclear receptors. Because of cross-talks between retinoids and VD3 pathways, we used NB4-LR2 cells in which RAR is knock-down. In this cell line, NCoA4 is expressed in a VDR-dependent fashion reinforcing the hypothesis that the coregulator is specifically involved in the VD3-monocytic differentiation of leukemic cells. Next, to explore the role of NCoA4, U937 cells were stably transfected to constitutively over-express the protein. The doubling time of this cell line (U-NCoA4) reaches to 48 hours against 24 hours in U937 cells. Concerning ligand-induced growth arrest, these cells are particularly sensitive to the RXR and VDR agonists while no significative difference was observed after treatment by the RAR agonist or by any combination. In addition, over-expression of NCoA4 induces a slow down of differentiation, as shown by expression of CD11b and CD14 myelomonocytic markers. Thus, in U-NCoA4, except for the RAR agonist, treatment for 72 hours corresponds to treatment for 48 hours levels of U937 cells. To conclude, in term of growth arrest, NCoA4 over-expressing cells are particularly sensitive to RXR and VDR agonists. Thus, natural ligands present in the culture medium might reduce or delay proliferation, inducing the same effect on differentiation. In order to have a large view of networks, transcriptome of U-NCoA4 was analyzed by real time PCR on Low Density Array composed of a hundred messengers extracted from U937 SAGE libraries. Analysis is currently in progress.

It is now well established that all trans Retinoic Acid (atRA), administered at pharmacological doses to Acute Promyelocytic Leukemia (APL) patients, provides the first example of therapy by differentiation. Clinical remission is often transient as resistance develops. Because mechanisms of installation are still unclear, gene expression changes during APL cells differentiation were identified by Serial Analysis of Gene Expression (SAGE). Construction of proliferative and 48 hours atRA-treated NB4 cells libraries allowed us to identify a set of new transcriptional markers. Expression profiles of atRA response were performed on NB4, two atRA-resistant cell lines (NB4-LR2 and UF1) and APL blasts by real time PCR analyze on Microfluidic Card. We choose a hundred genes from multiple functional classes since there are several potential mechanisms for atRA resistance: target gene expression, transcription regulation, atRA metabolism, proteasome pathways≡ Reliability between SAGE and Microfluidic technologies is high since 95% of significantly modulated SAGE transcripts (p Transcriptome studies were conducted onto blast of patients with distinct long-term sensitivity, established by correlation to the in vitro differentiation rate (Cassinat, B. et al. Blood, 2001). As a result, all patients show transcriptional response to retinoid. However, once blast differentiation reached, induction of atRA-response element genes in high sensitive blasts is reduced. In opposition, transcripts expression of low sensitive patients is still high, revealing a delay in differentiation establishment. Conversely, expression of cytochrome p450 CYP26, involved in the atRA catabolism, is maintained in highly sensitive blast whereas no modulation is observed in low sensitive blast. Because promoter analysis reveals Homeobox response element, involvement of HOX factors found in SAGE librairies was investigated. In cell lines, HOX factors cooperate with retinoid receptors to increase CYP26 transcription. Furthermore, high-pressure chromatography shows a switch between atRA and its metabolites, 6 hours after atRA addition but only in sensitive cell line. To conclude, CYP26 is a relevant marker of resistance prognostic. Since metabolites show similar efficiency for cell growth inhibition and differentiation than atRA, their implication in resistance installation is investigated.

Calf fetuin, one of the 3 major known fetal proteins has been isolated by a two-step purification procedure and characterized by aminoacid composition. The purified glycoprotein, which consisted of a single chain, was submitted to 47 steps of automatic aminoacid sequencing, allowing to determine 44 positions. This section of the molecule was devoid of carbohydrates. Comparison of this sequence with a variety of detectable potentially related protein did not allow to point to any detectable homology.

Summary The treatment of lamb fetuin by Pronase is described. After purification by chromatography on Sephadex G 50, the pool of glycopeptides is fractionated and analysed by paper electrophoresis. By a specific coloration, two groups of glycopeptides are revealed and tentatively separated by DEAE-Cellulose chromatography. The chemical composition of theses glycopeptides is determined and their molecular weight estimated. From these results, its appears that lamb fetuin contains 3 carbohydrate units (M.W. = 3120), attached to the peptide chain by an N-glycosidic linkage and two other units containing no more than three osides, attached by an 0-glycosidic linkage involving GalNAc and the hydroxyl group of a Serine.

Resume L'AFP bovine avait ete signalee dans le serum foetal bovin. Ce travail decrit son identification et son isolement par chromatographie sur carboxymethylcellulose DEAE Sephadex A-50, gel de filtration et colonne d'immunoadsorbant. L'AFP se revele homogene, par electrophorese en gel d'acrylamide dans des conditions denaturantes et non denaturantes, par ultracentrifugation et en immunoelectrophorese. Les resultats suivants ont ete obtenus: o 1. La sedimentation a l'equilibre indique un poids moleculaire de 66 500 pour une constante de Svedberg s o 20, w = 4,71 s, un volume partiel specifique v = 0,737 ml g −1 est tire des mesures de densites. 2. L'electrophorese en gel de polyacrylamide SDS permet d'estimer le poids moleculaire a 67 000. 3. La microheterogeneite est demontree par electrofocalisation. 4. La composition chimique a ete determinee. Le point isoelectrique du variant principal est de 4,6. L'AFP est une glycoproteine contenant 7 p. cent de sucres : 1,67 p. cent d'hexoses, 2,38 p. cent de N -acetyl glucosamine, et 1,8 p. cent d'acide N -acetyl neuraminique. D'apres ces donnees un rayon de Stokes de 3,26 nm, un coefficient de diffusion D 20, w = 6,61 10 −7 cm 2 sec −1 et un coefficient de friction f/ fo = 1,21 ont ete calcules.

We have analysed Con A-stimulated mouse lymphocytes for the presence of 2′-5′-linked oligoadenylates using a radioimmunoassay based on a monoclonal antibody raised against adenylyl (2′-5′) adenosine (A2′pA). Time-course and Con A dose dependence were performed. We found that Con A induced, in a dose-dependant manner, the accumulation of immunoreactive material, together with the incorporation of 3H-thymidine in DNA. We showed that the immunoreactive material was constituted for the essential, by a novel 2′-5′ oligoadenylate. It was isolated and characterized as adenylyl 2′-5′adenylic acid (2′ and 3′P) according to the combination of criteria such as immunoreactivity, enzyme susceptibility, chromatographic behaviour and comparison with A2′pA3′(32P)p, A2′pA3′p acid A2′pA2′p that we have chemically synthetized. This is the first example of significant variations of a 2′-5′ oligoadenylate level in circumstances other than the antiviral mechanism of interferon.

Monoclonal antibodies directed against 2'-5' oligoadenylates (pxA(2'pA)n 0 less than or equal to x less than or equal to 3, n greater than or equal to 1) have been obtained with A2'pA-succinyl albumin as immunogen. A competition assay using 125I iodo succinyl A2'pA tyrosine methyl ester as a tracer and thirty chemically related analogs was used to investigate the molecular basis governing their reactivity. We show that the use of a hapten as small as A2'pA elicits antibodies of high affinity for this dinucleotide (Kd = 2 x 10(-11) M). The overall immunoreactivity is essentially shared between the first moiety (5'OH A2'p) and the 2'-5' phosphodiester bond; however, the second moiety is an integral part of the epitope which extends up to the spacer. Therefore, any modification at the 5'OH end or of the 2'-5' structure dramatically decreases the binding. Modifications at the 2' and/or 3' ends are favourable if they mimic the immunogen. Modifications of the ribose backbone reveal the part of antigenicity due to the different 3'OH groups. Substitution of the bases show that the second adenine is implicated in the antigenicity. We demonstrate how, with such requirements, these antibodies recognize A2'pA alone or at the 5' end of or else included in longer oligonucleotides.

Haptoglobin (Hp) is a glycoprotein which is found in the serum of several animals. Its characteristic property is the ability to form with hemoglobin (Hb) a stable equimolecular complex Hp-Hb which Possess a peroxidase activity; this property allows a quantitative estimation of Hp [ 1 ] . The normal level of Hp varies according to the species: from 0.05 g/l in Bovidae and Cervidae, to 1.2 g/l in human. It always rises in any inflammatory state [2]. In the sheep, Hp is composed of a series of polymeric proteins, whose electrophoretic pattern is very similar to that of human Hp 2-2 [3]. Their mol. wts. range from lo5 to 10” [3]. They are formed in the human by combination in different numbers of two polypeptidic chains called (Y and 0 [4]. We have undertaken the purification of sheep Hp in order to study its structure. We have controlled the purification by using the Hp estimation after each stage. But, as we shall establish later, sheep Hp is easily altered: below pH 4 or above pH 6.5, it looses its ability to combine with Hb. For these reasons, the preparation of this protein in the native state is very difficult. Therefore, after two successive chromatographic purifications, we have used an immunological technique as a last step of purification. The method is based on the following fact: since the Hp level in normal sheep is very low, a rabbit immune-serum anti-normal-sheep serum contains a very small quantity of y-globulins anti-haptoglobin. After insolubilisation of these rabbit antibodies, affinity chromatography retains selectively proteins

A radioimmunoassay for fetuin is described. It uses [125I]fetuin and rabbit antisera. The bound antigens are precipitated by anti-rabbit globulins coupled to Sepharose. This assay has been used to compare the immunological properties of ovine and bovine fetuin. It allows to detect a serum antigen cross-reacting with fetuin in pig species. A very faint reaction is observed with human fetal serum, that does not support the existence of a fetuin like antigen in human.

Summary Evidence for microheterogeneity of lamb fetuin was obtained by electrophoresis on different supports of either purified fetuin or total fetal serum. In both cases, 7–8 bands were observed corresponding to the different variants. Fetuin electrophoretic mobility slightly increases during the first two months of pregnancy and then remains constant from the 3d month until birth. During this later stage, the pHi of all the variants were found between 3.88 and 4.28 as determined by electrofocusing. Seven fractions can be resolved by CM-cellulose chromatography, each generally containing a major variant along with 2 minor ones. In all fractions, the hexose and osamine content remains constant while that of NANA gradually decreases from fraction 1 to fraction 7. However, the difference in NANA content between fraction 1 and fraction 7 does not exceed two residues per molecule and therefore cannot account for the number of variants. After neuraminidase treatment of each individual fraction, 5 to 6 bands can be observed by electrophoresis at pH 5.5. When the same treatment is applied to unfractionated purified fetuin, 7 to 8 bands are still observed. From the above results, we conclude that the variations in the carbohydrate content of lamb fetuin are insufficient to account for its microheterogeneity. We therefore postulate that structural isomerisms might also be involved in this phenomenon.