ABSTRACT
The response of wild fish to pollutants was studied using two biomarkers in chub (Leuciscus cephalus) at five stations in the Moselle River (France) in 1998 and in 1999. The induction of cytochrome P450 1A was quantified by the ethoxyresorufin O-deethylase (EROD) activity in the liver and the level of DNA single-strand breaks was determined in erythrocytes using the comet assay. EROD activity was observed to be up to 10-fold induced in both males and females from the downstream stations in comparison to the fish from the upstream station. Levels of DNA damage did not parallel EROD induction. Chemical analyses did not clearly explain the responses of the studied biomarkers, confirming the great difficulty in relating chemical and biological information in the field. This study confirms the difficulty in assessing the biological effects of mixtures of pollutants and points out the usefulness of a large array of biomarkers.
Subject(s)
Cyprinidae/genetics , Cyprinidae/metabolism , Cytochrome P-450 CYP1A1/biosynthesis , DNA Damage , Erythrocytes/drug effects , Liver/drug effects , Water Pollutants, Chemical/toxicity , Animals , Biomarkers , Comet Assay , Cyprinidae/growth & development , Erythrocytes/ultrastructure , Female , France , Liver/enzymology , Male , Muscles/chemistry , Water Pollutants, Chemical/pharmacokineticsABSTRACT
My contribution to this special issue on Vertebrate Sex Chromosomes deals with the theme of X chromosome inactivation and its variations. I will argue that the single active X--characteristic of mammalian X dosage compensation--is unique to mammals, and that the major underlying mechanism(s) must be the same for most of them. The variable features reflect modifications that do not interfere with the basic theme. These variations were acquired during mammalian evolution--to solve special needs for imprinting and locking in the inactive state. Some of the adaptations reinforce the basic theme, and were needed because of species differences in the timing of interacting developmental events. Elucidating the molecular basis for the single active X requires that we distinguish the mechanisms essential for the basic theme from those responsible for its variations.
Subject(s)
Dosage Compensation, Genetic , X Chromosome/genetics , Animals , Evolution, Molecular , Genetic Variation , Humans , RNA, Long Noncoding , RNA, Untranslated/geneticsABSTRACT
X inactivation is the mammalian method for X-chromosome dosage compensation, but some features of this developmental process vary among mammals. Such species variations provide insights into the essential components of the pathway. Tsix encodes a transcript antisense to the murine Xist transcript and is expressed in the mouse embryo only during the initial stages of X inactivation; it has been shown to play a role in imprinted X inactivation in the mouse placenta. We have identified its counterpart within the human X inactivation center (XIC). Human TSIX produces a >30-kb transcript that is expressed only in cells of fetal origin; it is expressed from human XIC transgenes in mouse embryonic stem cells and from human embryoid-body-derived cells, but not from human adult somatic cells. Differences in the structure of human and murine genes indicate that human TSIX was truncated during evolution. These differences could explain the fact that X inactivation is not imprinted in human placenta, and they raise questions about the role of TSIX in random X inactivation.
Subject(s)
Dosage Compensation, Genetic , RNA, Antisense/genetics , RNA, Untranslated/genetics , Transcription Factors/genetics , Aging/genetics , Animals , Cell Line , Embryo, Mammalian/metabolism , Evolution, Molecular , Fetus/metabolism , Genomic Imprinting/genetics , Humans , Mice , Molecular Sequence Data , Open Reading Frames/genetics , Placenta/metabolism , RNA, Antisense/analysis , RNA, Antisense/biosynthesis , RNA, Antisense/isolation & purification , RNA, Long Noncoding , RNA, Untranslated/analysis , RNA, Untranslated/biosynthesis , RNA, Untranslated/isolation & purification , Sequence Deletion/genetics , Sequence Homology, Nucleic Acid , Species Specificity , Stem Cells/metabolism , Transcription Initiation Site , Transcription, Genetic , Transgenes/geneticsABSTRACT
X chromosome inactivation is initiated from a segment of the mammalian X chromosome called the X inactivation center. Transgenes from this region of the murine X chromosome are providing the means to identify the DNA needed for cis inactivation in mice. We recently showed that chimeric mice carrying transgenes from the human X inactivation center (XIC) region also provide a functional assay for human XIC activity; approximately 6 copies of a 480-kb human transgene (ES-10) were sufficient to initiate random X inactivation in cells of male chimeric mice (Migeon et al., 1999, Genomics, 59, 113-121). Now, we report studies of another human transgene (ES-5), which contains less than 300 kb of the human XIC region on Xq13.2 including an intact XIST locus and which has inserted in one or two copies into mouse chromosome 6. The ES-5 transgene is recognized as an X inactivation center in mouse embryonic stem cells, but is not sufficient to induce random X inactivation in somatic cells of highly chimeric mice. Human transgenes in chimeric mice provide a means to uncouple the key steps in this complex pathway and facilitate the search for essential components of the human XIC region.
Subject(s)
Dosage Compensation, Genetic , Gene Dosage , Transgenes/genetics , Animals , Cells, Cultured , Chimera/genetics , Clone Cells , Embryo, Mammalian/cytology , Female , Fetal Death/genetics , Humans , Male , Mice , Mice, Transgenic , RNA/metabolism , RNA, Long Noncoding , RNA, Untranslated/genetics , Stem Cells/metabolism , Transcription Factors/genetics , TransfectionABSTRACT
We report the second trimester prenatal echographic diagnosis of an epignathus teratoma in a female fetus with ring X chromosome mosaicism. The ring X chromosome mosaicism was present in the amniotic cell culture and in the teratoma and the ring X was inactive (X-inactive specific transcript (XIST) locus expressed). Hypoplastic left heart with valvular aortic stenosis and non-immune hydrops were additional findings, and are well-documented in Turner syndrome. The occurrence of epignathus teratoma in Turner syndrome has not been documented sofar.
Subject(s)
Fetal Diseases/diagnosis , Nasopharyngeal Neoplasms/diagnosis , Ring Chromosomes , Sex Chromosome Aberrations , Teratoma/diagnosis , Turner Syndrome/complications , X Chromosome , Amniocentesis , Female , Fetal Diseases/genetics , Humans , Mosaicism/genetics , Nasopharyngeal Neoplasms/genetics , Pregnancy , Pregnancy Trimester, Second , Teratoma/genetics , Ultrasonography, PrenatalABSTRACT
Mental retardation and congenital malformations in individuals with small ring X chromosomes are often due to the functional disomy that results from failure of these chromosomes to undergo X inactivation. Such chromosomes either lack the XIST locus or do not express it. We have carried out genetic analysis of the ring X chromosomes from two girls with a 45,X/46,X,r(X) karyotype, mental retardation, and a constellation of abnormalities characteristic of the severe phenotype due to X disomy. In each case the ring X chromosome included an intact XIST locus which was expressed; the breakpoints were distal to DXS128, and therefore outside the XIC region; transcription analysis of alleles at the androgen receptor locus confirmed that these were inactive chromosomes. The characteristics of the XIST RNA were similar to the wild-type. Additional studies in cultured fibroblasts showed a second ring in a small percentage of the cells. The association of severe phenotype with an inactive X chromosome most likely reflects the presence of a second ring X chromosome which was active at least in some tissues during embryogenesis, but is no longer prominent in the tissues we analyzed.
Subject(s)
Dosage Compensation, Genetic , RNA, Untranslated , Ring Chromosomes , X Chromosome , Female , Humans , Infant , RNA, Long Noncoding , RNA, Messenger/genetics , Receptors, Androgen/genetics , Transcription Factors/geneticsABSTRACT
A two-step purification protocol was developed to purify rainbow trout (Oncorhynchus mykiss) vitellogenin (Vtg) and was successfully applied to Vtg of chub (Leuciscus cephalus) and gudgeon (Gobio gobio). Capture and intermediate purification were performed by anion-exchange chromatography on a Resource Q column and a polishing step was performed by gel permeation chromatography on Superdex 200 column. This method is a rapid two-step purification procedure that gave a pure solution of Vtg as assessed by silver staining electrophoresis and immunochemical characterisation.
Subject(s)
Vitellogenins/isolation & purification , Animals , Blotting, Western , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Fishes , Species Specificity , Spectrophotometry, Ultraviolet , Vitellogenins/biosynthesisABSTRACT
X chromosome inactivation is the means to downregulate the transcriptional output of X chromosomes in female mammals. Essential DNA from the murine X inactivation center (Xic) has been identified by introducing it into male embryonic stem (ES) cells. To identify the essential sequences on human X chromosomes, we transfected male mouse ES cells with a YAC transgene containing 480 kb of the putative human X inactivation center (XIC). Despite little DNA sequence conservation, the human transgene is recognized as a second Xic in these XY mouse cells and induces random inactivation in chimeric mice derived from these cells. Inactivation is extensive on the X chromosome, but more localized on chromosome 11 carrying the transgene, demonstrating that initial inactivation and spreading of inactivation signals along the chromosome are independent events. Our results show for the first time that the DNA included in the human XIC transgene is sufficient to initiate random X inactivation, even in cells of another species. Interspecies XIC trangenes should facilitate further investigation of this process in humans and other mammals.
Subject(s)
Dosage Compensation, Genetic , RNA, Untranslated , Transcription Factors/genetics , X Chromosome/genetics , Animals , Chimera , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Humans , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , RNA, Long Noncoding , Stem Cells/cytology , Stem Cells/metabolism , Transfection , Transgenes/geneticsABSTRACT
The impact of a wastewater treatment plant (WWTP) effluent was assessed with the fish biomarker ethoxyresorufin-O-deethylase (EROD) using field and on-site laboratory experiments. EROD activity was measured in chub (Leuciscus cephalus) and stone loach (Noemacheilus barbatulus) caught at three sites of the Chalaronne River (southeast France). Liver somatic index (LSI) and organochloride bioaccumulation in muscle were estimated for chub only. In September, EROD activity and LSI of chub increased significantly between the sites above and below the WWTP effluent discharge. EROD induction detected in chub was confirmed by on-site tank experiments. EROD levels were determined in juvenile rainbow trout (Oncorhynchus mykiss) and mirror carp (Cyprinus carpio) exposed to different concentrations of the WWTP effluent and river water for 16 days. After a 4-day exposure, EROD activities of the carp exposed to the effluent increased significantly compared with the control. The response was linked to the effluent concentration and was stable with exposure time. WWTP effluent induced EROD activity, whereas organic and metal analyses, performed on fish muscle and sediment, did not indicate any difference between upstream and downstream of the discharge.
Subject(s)
Cyprinidae/metabolism , Cytochrome P-450 CYP1A1/analysis , Environmental Monitoring/methods , Oncorhynchus mykiss/metabolism , Sewage/chemistry , Waste Management , Animals , Female , France , Fresh Water , Liver/metabolism , Male , Muscles/metabolism , Seasons , Sewage/analysisABSTRACT
The aim of the study is to investigate the potential of a feedforward neural network for detecting wavelet preprocessed late potentials. The terminal parts of a simulated QRS complex are processed with a continuous wavelet transform, which leads to a time-frequency representation of the QRS complex. Then, diagnostic feature vectors are obtained by subdividing the representations into several regions and by processing the sum of the decomposition coefficients belonging to each region. The neural network is trained with these feature vectors. Simulated ECGs with varying signal-to-noise ratios are used to train and test the classifier. Results show that correct classification ranges from 79% (high-level noise) to 99% (no noise). The study shows the potential of neural networks for the classification of late potentials that have been preprocessed by a wavelet transform. However, clinical use of this method still requires further investigation.
Subject(s)
Arrhythmias, Cardiac/diagnosis , Electrocardiography , Neural Networks, Computer , HumansABSTRACT
A salient feature of mammalian X dosage compensation is that X-inactivation occurs without regard to the parental origin of either active or inactive X. However, there are variations on the theme of random inactivation, namely paternal X inactivation in marsupials and in placental tissues of some mammals. Whether inactivation is random or paternal seems to depend on the time when this developmental program is initiated. As deletions of the X inactivation center (XIC/Xic) and/or the X inactive specific transcript (XIST/Xist) gene result in failure of cis X-inactivation, mutations in genes from this region might lead to preferential inactivation of one X chromosome or the other. The Xce locus in the murine Xic is considered a prototype for this model. Recent studies suggest that choice involves maintaining the activity of one X, while the other(s) by default is programmed to become inactive. Also, choice resides within the XIC, so that mutations elsewhere, although perhaps able to interfere with cis inactivation, are not likely to affect the X chromosome from only one parent. Mutations affecting the choice of active X will be more difficult to detect in humans than in inbred laboratory mice because of the greater allelic differences between maternal and paternal X chromosomes; some of these differences predispose to growth competition between the mosaic cell populations. I suggest that the skewing of inactivation patterns observed in human females most often occurs after random X inactivation, and is due mainly to cell selection favoring alleles that provide a relative growth advantage.
Subject(s)
Dosage Compensation, Genetic , Alleles , Animals , Cell Division , Gene Deletion , Humans , Mammals , Mice , Mutation , Species Specificity , X ChromosomeABSTRACT
A comparison of ethoxyresorufin-O-deethylase (EROD) data collected in 1995 in various sites in the Rhône watershed (France) was carried out to quantify the influence of factors such as contamination and biological parameters on EROD levels and within-group variabilities. Three species of cyprinids were collected and fish chemical contamination was measured. A log transformation of EROD data provided both normalization and homogeneity of variances. The influence of female sexual maturation on the variability and EROD dimorphism was quantified. A relationship with contaminant bioaccumulation was observed. A comparison with EROD data collected during previous studies by the same laboratory was made to validate the results.
Subject(s)
Cyprinidae/physiology , Cytochrome P-450 CYP1A1/metabolism , Environmental Monitoring/methods , Water Pollutants, Chemical/toxicity , Animals , Biomarkers , Cytochrome P-450 CYP1A1/analysis , France , Reproducibility of Results , Tissue Distribution , Water Pollutants, Chemical/pharmacokineticsSubject(s)
Cell Division/genetics , Dosage Compensation, Genetic , Genetic Linkage , X Chromosome , Female , Fetal Death/genetics , Gene Deletion , Humans , MaleABSTRACT
A de novo dicentric Y;21 (q11.23;p11) translocation chromosome with one of its two centromeres inactive has provided the opportunity to study the relationship between centromeric inactivation, the organization of alphoid satellite DNA and the distribution of CENP-C. The proband, a male with minor features of Down's syndrome, had a major cell line with 45 chromosomes including a single copy of the translocation chromosome, and a minor one with 46 chromosomes including two copies of the translocation chromosome and hence effectively trisomic for the long arm of chromosome 21. Centromeric activity as defined by the primary constriction was variable: in most cells with a single copy of the Y;21 chromosome, the Y centromere was inactive. In the cells with two copies, one copy had an active Y centromere (chromosome 21 centromere inactive) and the other had an inactive Y centromere (chromosome 21 centromere active). Three different partial deletions of the Y alphoid array were found in skin fibroblasts and one of these was also present in blood. Clones of single cell origin from fibroblast cultures were analysed both for their primary constriction and to characterise their alphoid array. The results indicate that (1) each clone showed a fixed pattern of centromeric activity; (2) the alphoid array size was stable within a clone; and (3) inactivation of the Y centromere was associated with both full-sized and deleted alphoid arrays. Selected clones were analysed with antibodies to CENP-C, and staining was undetectable at both intact and deleted arrays of the inactive Y centromeres. Thus centromeric inactivation appears to be largely an epigenetic event.
Subject(s)
Chromosomes, Human, Pair 21/genetics , Translocation, Genetic , Y Chromosome/genetics , Adolescent , Centromere/genetics , Centromere/metabolism , Centromere/ultrastructure , Chromosomal Proteins, Non-Histone/metabolism , Chromosomes, Human, Pair 21/metabolism , Chromosomes, Human, Pair 21/ultrastructure , Clone Cells , Cytogenetics , DNA, Satellite/genetics , Down Syndrome/genetics , Down Syndrome/metabolism , Humans , In Situ Hybridization, Fluorescence , Male , Phenotype , Y Chromosome/metabolism , Y Chromosome/ultrastructureABSTRACT
An echographic image processing method has been developed, and validated by in vitro experiments, for the 3D reconstruction of the long bones of the newborn. The reconstruction of successive parallel cross-sections is obtained by a 2D reconstruction technique using radial B-scan image processing. The automatic segmentation of all the calculated images allows the extraction of the external contours of the skeleton. After structuring the explored volume using a contour association method, a contour interpolation step is required to solve the anisotropy problem, to obtain a 3D representation with cubic voxel lists. The results are encouraging, and a new mechanical part prototype of the acquisition system is under test for in vivo experiments. The main originality of the paper lies in the combination of different steps to obtain a practical solution to a clinical problem.
Subject(s)
Bone and Bones/diagnostic imaging , Image Processing, Computer-Assisted/methods , Femur/diagnostic imaging , Humans , Infant, Newborn , Tibia/diagnostic imaging , UltrasonographyABSTRACT
We describe the cloning and characterization of the NTT gene (noncoding transcript in T cells), identified by differential display RT-PCR based on the differential presence of its transcript in a subset of activated, human CD4+ T-cell clones. The full-length cDNA and genomic sequences were cloned and found to produce a 17-kb transcript that is polyadenylated, but is not spliced. Consistent with the transcript's nuclear predominance, NTT has no open reading frame larger than 270 bp. It is transcribed in a select subset of CD4+ T-cell clones propagated in vitro. Its transcription can also be induced in freshly isolated T cells by in vitro activation with PHA or with PMA and ionomycin. In vivo, NTT transcripts are found only in activated, but not resting, T cells. Transcripts were absent in a variety of lymphoid cell lines and transformed lines from other tissues. NTT is a new member of the small group of genes including XIST (X-specific transcript), H19, and IPW (imprinted gene in the Prader-Willi syndrome region), which are transcribed but not translated, and may have a role in the regulation of neighboring genes. XIST, H19, and IPW exhibit monoallelic expression, but both NTT alleles are expressed in CD4+ T-cell clones. Southern blot and fluorescence in situ hybridization analyses show that NTT is a single-copy gene residing in chromosome 6q23-q24, near the interferon-gamma receptor gene (IFN-gamma R). Their proximity and shared expression pattern suggest a possible functional relationship.
Subject(s)
CD4-Positive T-Lymphocytes/metabolism , Lymphocyte Activation , RNA, Nuclear/genetics , Alleles , CD4-Positive T-Lymphocytes/immunology , Cell Line , Chromosome Mapping , Chromosomes, Human, Pair 6 , Cloning, Molecular , DNA, Complementary , Gene Expression , Genome, Human , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Lymphocyte Activation/genetics , Molecular Sequence Data , Poly A/metabolism , Polymerase Chain Reaction , RNA Splicing , RNA, Messenger/genetics , Repetitive Sequences, Nucleic Acid , Sequence Alignment , Transcription, GeneticSubject(s)
Cholinesterase Inhibitors/toxicity , Copper Sulfate/toxicity , Cytochrome P-450 CYP1A1/metabolism , Insecticides/toxicity , Organothiophosphorus Compounds/toxicity , Animals , Cholinesterase Inhibitors/administration & dosage , Copper Sulfate/administration & dosage , Environmental Exposure , Enzyme Induction/drug effects , Frozen Sections , Injections, Intraperitoneal , Insecticides/administration & dosage , Lethal Dose 50 , Liver/drug effects , Liver/enzymology , Muscles/drug effects , Muscles/enzymology , Oncorhynchus mykiss , Organothiophosphorus Compounds/administration & dosage , beta-Naphthoflavone/administration & dosage , beta-Naphthoflavone/toxicityABSTRACT
We have previously reported functional disomy for X-linked genes in females with tiny ring X chromosomes and a phenotype significantly more abnormal than Turner syndrome. In such cases the disomy results from failure of these X chromosomes to inactivate because they lack DNA sequences essential for cis X inactivation. Here we describe a novel molecular mechanism for functional X disomy that is associated with maternal isodisomy. In this case, the severe mental retardation and multiple congenital abnormalities in a female with a mosaic 45,X/ 46,X,del(X)(q21.3-qter)/ 46X,r(X) karyotype are associated with overexpression of the genes within Xpter to Xq21.31 in many of her cells. Her normal X, ring X, and deleted linear X chromosomes originate from the same maternal X chromosome, and all are transcriptionally active. None expresses X inactive specific transcript (XIST), although the locus and region of the putative X inactivation center (XIC) are present on both normal and linear deleted X chromosomes. To our knowledge, this is the first report of a functional maternal X isodisomy, and the largest X chromosome to escape inactivation. In addition, these results (1) show that cis inactivation does not invariably occur in human females with two X chromosomes, even when the XIC region is present on both of them; (2) provide evidence for a critical time prior to the visible onset of X inactivation in the embryo when decisions about X inactivation are made; and (3) support the hypothesis that the X chromosome counting mechanism involves chromosomal imprinting, occurs prior to the onset of random inactivation, and is required for subsequent inactivation of the chromosome.
Subject(s)
Chromosome Deletion , Embryonic and Fetal Development/genetics , Ploidies , Sex Chromosome Aberrations , Turner Syndrome/genetics , X Chromosome , Cell Division , Cells, Cultured , Child , Chromosome Mapping , DNA Replication , Female , Genetic Markers , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence , Karyotyping , Male , Mosaicism , Polymerase Chain Reaction , Pregnancy , Transcription, Genetic , Turner Syndrome/metabolismABSTRACT
We have recently reported results of DNA replication analysis of three X-linked loci (FRAXA, F8C and XIST) on the X chromosomes in male and female fibroblasts using fluorescence in situ hybridization (FISH) (1). Although our findings that XIST replicates later on the active X than on the inactive X are similar to those of Boggs & Chinault (2) based on a FISH assay in female lymphoblasts, they are the opposite of observations recently reported by Hansen et al. (3) using a different technique. Because our conclusions about the inactive X were deduced from the behavior of the active X in male cells, we reexamined the time when these loci replicate on the human inactive X chromosome isolated from its homolog in somatic cell hybrids. We also studied the same chromosome as an active X in related hybrids. The results provide direct evidence that the expressed XIST locus on the inactive X replicates earlier than its repressed homolog on the active X and earlier than the FRAXA locus which is repressed on this chromosome. The silent XIST locus on the active X replicates late along with F8C which is also not transcribed in these cells. Possible reasons for the different results obtained by Hansen et al. (3) are discussed.
Subject(s)
DNA Replication , Guanosine Diphosphate/genetics , RNA, Untranslated , Transcription Factors/genetics , X Chromosome , Animals , Cells, Cultured , Chromosome Mapping , Fibroblasts , Genetic Markers , Genetic Variation , Humans , Hybrid Cells , In Situ Hybridization, Fluorescence/methods , Male , Mice , RNA, Long Noncoding , Skin/cytology , Skin/metabolismABSTRACT
Small ring X chromosomes were first described in mosaic karyotypes of females with the relatively benign phenotype of Turner syndrome. The presence of these rings in association with more severe phenotypes including mental retardation has raised the possibility that they lack sequences necessary for X chromosome inactivation, specifically genes within the X inactivation center (XIC) essential for cis X-inactivation. We recently showed that ring X chromosomes ascertained because of the severe phenotype do not express XIST, a candidate for the relevant gene, and that they are in fact active chromosomes. We now report studies of the genetic content of 11 of these ring X chromosomes (9 associated with severe phenotypes). Our results indicate that these chromosomes contain contiguous segments of DNA and have variable proximal and distal breakpoints and some include mainly long arm or mainly short arm sequences. As expected for ring chromosomes, they lack telomeric sequences. Many of the ring chromosomes lack the XIST locus, consistent with XIST being necessary for cis inactivation. However, the breakpoints in four ring chromosomes that have XIST sequences but do not express XIST suggest that other sequences within the XIC distal to XIST as it is now defined are also needed.