Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activating cis-recombination events. A striking example is the T-cell receptor beta-chain (TCRbeta) gene enhancer (Ebeta), which in the mouse consists of at least seven nuclear factor binding motifs (betaE1 to betaE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Ebeta required for recombination enhancer activity. The Ebeta core is comprised of a limited set of motifs (betaE3 and betaE4) and an additional previously uncharacterized 20-bp sequence 3' of the betaE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the betaE1 through betaE4 motifs but not the 3' sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Ebeta recombinational activity. Our results emphasize the multifunctionality of the TCRbeta enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.

Characterization of tumor-specific transplantation antigen induced in cells infected or transformed by polyomavirus.

Tumor-specific transplantation antigen (TSTA) activity was investigated (with the use of inbred BALB/c and C3H mice and inbred Fisher and Wistar rats) in various early polyomavirus (Py)-coded proteins by three methodologies: 1) immunoprecipitation by anti-T-antigen sera, gel slicing, and immunization; 2) affinity chromatography with the use of anti-tumor-associated antigen serum; and 3) use of a Py mutant, NG18. The results obtained by all three techniques allowed refutation of the hypothesis that the three major species of T-antigen possess the main TSTA activity, particularly middle-sized T-antigen which is a plasma membrane protein. TSTA activity was found to be associated with proteins having a molecular weight of approximately 37,000. The exact origin of this protein is discussed.

cDNA sequence and chromosomal localization of the NDUFS8 human gene coding for the 23 kDa subunit of the mitochondrial complex I.

We have sequenced the cDNA for the 23 kDa subunit of the human mitochondrial respiratory complex I. The deduced protein consists of 210 amino acids (Mr = 23705 Da) with a 34 amino acid N terminus presumably acting as a presequence for mitochondrial import. The predicted mature protein (Mr = 20290 Da) is 92% identical to the bovine mitochondrial subunit and 72% to the Rhodobacter capsulatus NUOI counterpart. Two clusters of four cysteine residues are conserved among these proteins. The gene (NDUFS8) coding for the human subunit has been mapped to chromosome 11q13.

Disruption of a new X linked gene highly expressed in brain in a family with two mentally retarded males.

BACKGROUND: Mental retardation (MR) affects 2-3% of the human population and some of these cases are genetically determined. Although several genes responsible for MR have been identified, many cases have still not been explained. METHODS: We have identified a pericentric inversion of the X chromosome inv(X)(p22.3;q13.2) segregating in a family where two male carriers have severe MR while female carriers are not affected. RESULTS: The molecular characterisation of this inversion led us to identify two new genes which are disrupted by the breakpoints: KIAA2022 in Xq13.2 and P2RY8 in Xp22.3. These genes were not previously fully characterised in humans. KIAA2022 encodes a protein which lacks significant homology to any other known protein and is highly expressed in the brain. P2RY8 is a member of the purine nucleotide G-protein coupled receptor gene family. It is located in the pseudo-autosomal region of the X chromosome and is not expressed in brain. CONCLUSIONS: Because the haploinsufficiency of P2RY8 in carrier mothers does not have a phenotypic consequence, we propose that the severe MR of the affected males in this family is due to the absence of the KIAA2022 gene product. However, screening 20 probands from X linked MR families did not reveal mutations in KIAA2022. Nonetheless, the high expression of this gene in fetal brain and in the adult cerebral cortex could be consistent with a role in brain development and/or cognitive function.

ATR-X mutations cause impaired nuclear location and altered DNA binding properties of the XNP/ATR-X protein.

Mutations in the XNP/ATR-X gene, located in Xq13.3, are associated with several X linked mental retardation syndromes, the best known being alpha thalassaemia with mental retardation (ATR-X). The XNP/ATR-X protein belongs to the family of SWI/SNF DNA helicases and contains three C2-C2 type zinc fingers of unknown function. Previous studies have shown that 65% of mutations of XNP have been found within the zinc finger domain (encoded by exons 7, 8, and the beginning of exon 9) while 35% of the mutations have been found in the helicase domain extending over 3 kb at the C-terminus of the protein. Although different types of mutations have been identified, no specific genotype-phenotype correlation has been found, suggesting that gene alteration leads to a loss of function irrespective of mutation type. Our aims were to understand the function of the XNP/ATR-X protein better, with specific attention to the functional consequences of mutations to the zinc finger domain. We used monoclonal antibodies directed against the XNP/ATR-X protein and performed immunocytochemical and western blot analyses, which showed altered or absent XNP/ATR-X expression in cells of affected patients. In addition, we used in vitro experiments to show that the zinc finger domain can mediate double stranded DNA binding and found that the DNA binding capacity of mutant forms in ATR-X patients is severely reduced. These data provide insights into the understanding of the functional significance of XNP/ATR-X mutations.

Abnormal expression of the KLF8 (ZNF741) gene in a female patient with an X;autosome translocation t(X;21)(p11.2;q22.3) and non-syndromic mental retardation.

Non-syndromic X linked mental retardation (MRX) is a heterogeneous group of conditions in which all patients have mental retardation as the only constant phenotypic feature. We have identified a female patient with mental retardation and a balanced translocation involving chromosomes X and 21, t(X;21)(p11.2;q22.3). Physical mapping of the translocation breakpoint on the human X chromosome was performed using fluorescence in situ hybridisation. We have mapped the X chromosome breakpoint to a 21 kb DNA fragment upstream of the first exon of the KLF8 (ZNF741) gene in Xp11.21. We have subsequently shown that the KLF8 transcript is no longer detected in cells from the patient, although KLF8 expression is otherwise normally present in control lymphoblasts. Mutation screening of probands from 20 unrelated XLMR families linked to the proximal short arm of the human X chromosome failed to show any mutation in the coding region of the KLF8 gene.

Clinical heterogeneity in 16 patients with inv dup 15 chromosome: cytogenetic and molecular studies, search for an imprinting effect.

We report on clinical, cytogenetic and molecular analyses of 16 patients with inv dup (15) chromosome. We define the content of the inv dup (15) markers, their meiotic origin and the methylation status of the chromosome region involved. Precise phenotype-karyotype-genotype correlations allowed the identification of five different types of marker and demonstrated that even when the molecular content of the inv dup (15) chromosome clearly contributes to the severity of the phenotype, it does not appear to be the only relevant factor. All the markers were of maternal origin with an identical methylation profile, and neither imprinting nor methylation can explain the phenotypic variability. We suggest that the degree of phenotypic severity may be correlated with the severity of epilepsy.

Thyrotrophin regulation of apical and basal exocytosis of thyroglobulin by porcine thyroid monolayers.

Exocytosis, the ultimate step in thyroglobulin secretion, has been studied in porcine thyroid cells cultured in monolayers on the permeable bottom of culture chambers. We have previously demonstrated, using this culture system, that apical secretion accounts for 85-95% of total secretion of newly synthesized thyroglobulin. When cells were cultured for several days with bovine TSH (25 microU/ml) in the basal medium, the rate of glycoprotein accumulation in the upper compartment was three times higher than that in the absence of TSH. In contrast, the rate of thyroglobulin released into the basal medium (5-15% of total secreted thyroglobulin) appeared unmodified by chronic TSH stimulation. To investigate the effect of acute TSH stimulation on thyroglobulin exocytosis in the apical and basal compartments, pulse-chase experiments were carried out with the same culture system. The release of radiolabelled thyroglobulin (1.5-h pulse) into the apical medium was increased threefold during the 2-h chase period under TSH stimulation. The radiolabelled thyroglobulin released into the basal medium was increased only 1.5- to 2-fold, and stimulation disappeared after 1 h. The effect of TSH was maximal when the chase medium contained 50 microU TSH/ml. However, cells cultured for several days in the presence of 25 microU TSH/ml before the pulse-chase experiment, appeared desensitized to acute TSH stimulation. Similar responses were observed when the chase medium contained 8-chloro-cyclic AMP or cholera toxin. This study provides another example of the pleiotropic effect of TSH, mediated by cyclic AMP, on the sequential steps of thyroglobulin gene expression in cultured thyroid cells in which the polar character of the epithelial cells is well preserved.

Evaluation of the genotoxic activity of paclitaxel by the in vitro micronucleus test in combination with fluorescent in situ hybridization of a DNA centromeric probe and the alkaline single cell gel electrophoresis technique (comet assay) in human T-lymphocytes.

Paclitaxel is a recent chemotherapeutic agent that inhibits tubulin depolymerization in tumoral cells. Despite its increasing use against various human cancers, the genotoxicity of paclitaxel has never been studied on normal human cells. The in vitro genotoxic effects of the drug were evaluated with two complementary mutagenesis tests on human T-lymphocytes: (1) the cytokinesis-blocked micronuclei assay (CBMN) in combination with fluorescent in situ hybridization (FISH) of nonspecific centromeric probes and (2) the comet assay performed in three ways: on stimulated lymphocytes as in the CBMN, and on freshly isolated lymphocytes at both 4 and 37 degrees C. A slight cytotoxicity of 2.5 to 10 nM paclitaxel was found in the CBMN and a significant increase in the binucleated micronucleated cell rates was observed, with a concentration-dependent manner. In the FISH analysis, more than 85% of the micronuclei (MN) were centromere positive, and a ratio of 72. 2 to 78.6% of these MN contained more than one centromere. Moreover, at 10 nM of paclitaxel, 35.6% of the cells are multimicronucleated lymphocytes. Unexpectedly, paclitaxel induced single-strand breaks on proliferating lymphocytes at 5 and 7.5 nM but not in resting cells, even at 5 to 15 microM. These in vitro results showed that (1) paclitaxel does not present any direct DNA action in resting cells, (2) DNA damage detected in stimulated lymphocytes may be linked either to a high frequency of cells in the S-phase cell cycle or to a direct DNA damaging effect on replicating cells, and (3) paclitaxel is a strong in vitro aneugenic drug on human normal cells, at clinically relevant concentrations.

Immunolabelling and flow cytometry as new tools to explore dysferlinopathies.

Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, which lead to a reduced amount or a complete lack of dysferlin. One step in dysferlinopathies diagnosis consists in Western blot analysis of proteins extracted from muscle biopsy, or blood monocytes. We have taken advantage of dysferlin expression in monocytes to develop a whole blood flow cytometry (WBFC), using antibodies directed against dysferlin. Six patients were submitted to WBFC analysis and immunofluorescence analysis on monocytes. Results obtained are correlated to Western blot from monocytes and muscle biopsies. The possible usefulness of this flow cytometry analysis in routine diagnosis is presented.

Hormonal regulation of some steps of thyroglobulin synthesis and secretion in bicameral cell culture.

Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum-free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of 3H-mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive.

Chimpanzee Rh-like blood group genes map to chromosome region 1p36.1-->p34.2 by in situ hybridization.

A human Rh cDNA probe was used to map the Rh-like genes in the chimpanzee. The data gathered made it possible to uniquely localize these genes to chimpanzee chromosome region 1p36.1-->p34.2. This chromosomal localization is homologous to the location of the Rh genes in the human genome.

Bivalent 15 regularly associates with the sex vesicle in normal male meiosis.

Using fluorescent in-situ hybridization, we investigated the positioning of different human bivalents at the pachytene stage of normal male meiosis. We showed that, in about 35% of nuclei, the pericentromeric region of bivalent 15 is closely associated with the sex vesicle (SV). This behaviour may be linked to the presence of three domains in the pericentromeric region of chromosome 15: a large imprinted domain, a nucleolar organizing region (NOR), and a heterochromatic block. In order to define the domains of chromosome 15 involved in this association, we analysed the meiotic behaviour of other bivalents with similar domains: human bivalent 11 and mouse bivalent 7, bearing imprinted domains, other human acrocentric bivalents bearing a NOR, and the human bivalents 1, 9 and 16 containing a heterochromatic region. None of these bivalents were as frequently associated with the SV as the human bivalent 15. Nevertheless, we suggest that the bivalent 15 heterochromatin may be responsible for the association because of two properties: its telomeric location on chromosome 15 and its strong sequence homology with the Yq heterochromatin. This phenomenon could explain the high frequency of translocations between the chromosome 15 and the X or Y chromosomes.

HP1beta and HP1gamma, but not HP1alpha, decorate the entire XY body during human male meiosis.

During meiosis in male mammals, the X and Y chromosomes become heterochromatic and transcriptionally silent, and form the XY body. Although the HP1 proteins are known to be involved in the packaging of chromosomal DNA into repressive heterochromatin domains, their involvement in facultative heterochromatinization has not been precisely determined. Here, we analyse, for the first time in humans, the subcellular distribution of the heterochromatin protein HP1alpha, HP1beta and HP1gamma isoforms, in male pachytene spermatocytes, and the XY body facultative heterochromatin in particular. Our results demonstrate that HP1beta and HP1gamma, but not the HP1alpha isoforms, decorate the entire XY body in half the pachytene nuclei observed. In some nuclei, the XY body appears to be only partially labelled. In these cases, the HP1beta and HP1gamma signals are adjacent to the Yq12 constitutive heterochromatin and signal appears to originate in this region before spreading over the entire XY body. This distribution suggests that HP1beta and HP1gamma proteins, which are components of the constitutive heterochromatin, may also be involved in the facultative heterochromatinization of the XY body. Nevertheless, their absence from the early pachytene substage, even though the XY body is already condensed, suggests that these proteins are not involved in the initiation of this process.

In situ hybridization localizes the human OTF3 to chromosome 6p21.3-->p22 and OTF3L to 12p13.

Otf-3 (Octamer-binding transcription factor 3) is an octamer binding protein encoded by the murine gene Otf-3. Otf-3 belongs to a multigenic family and maps to the mouse Chromosome 17 between the Q and T regions within the major histocompatibility complex (MHC). We report the mapping of the human homologue: OTF3, to human chromosome 6p21.3-->p22 within or close to the human MHC class I region. Furthermore, one OTF3-like copy (OTF3L) is localized to 12p13.

Localization of ribosomal genes in human spermatogonia by fluorescent in situ hybridization and confocal microscopy.

The distribution of ribosomal RNA genes in nucleoli is still a matter of controversy. We have investigated the nucleolus of human type A spermatogonia, which displays a single, large fibrillar center. Silver-staining was used to localize the fibrillar center by light microscopy. Fluorescent in situ hybridization was performed on the same cell, using a 5.8-kb probe specific for a transcribed region of the ribosomal genes. The fluorescent area exactly corresponded to the silver-stained area. The three-dimensional distribution of rDNA was studied in horizontal and orthogonal sections using a confocal laser scanning microscope. The presence of the fluorescent signal throughout the whole silver-stained structure demonstrated that the fibrillar center, and at least a part of the dense fibrillar component, contained most of the rDNA.