Protein encoding by both DNA strands.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
F Mongelard
Publications and source records attributed to F Mongelard.
Two insulators are not better than one.
Explore the source record for details and available documents.
Animals↗
Genome organization in the human sperm nucleus studied by FISH and confocal microscopy.
The sperm nucleus has a unique chromatin structure where the DNA is highly condensed and associated with specific proteins, the protamines. It is a nondividing cell which is also transcriptionally inactive. After fusion with an oocyte, the sperm nucleus undergoes decondensation and, in the same time, starts replication and transcription. It has been suggested that somatic chromosomes during interphase are organized in territories which display a cell type and cell cycle specific distribution. The purpose of this work was to investigate whether chromosomes would also have a specific distribution in the sperm nucleus, which could be related to its inactive state, and have implications on the early stages of fertilization. In the present study, centromeric and telomeric sequences were detected by fluorescent techniques performed on human decondensed spermatozoa. Chromosome painting probes were used to detect the chromosome X and chromosome 13 on interphase sperm nuclei. The fluorescent signals were captured in 3D with a confocal microscope. For each of these chromatin structures, the volume, position, and distribution of the signals were analyzed in samples of 30 nuclei with the help of image analysis software. The centromeres appeared grouped in several foci that were randomly distributed within the sperm nucleus. The telomeres gave an approximately haploid number of small signals, evenly distributed throughout the nucleus. The chromosomes X and 13 occupied 4.7% and 3. 7% of the total nuclear volume, respectively. Interestingly, the X chromosome territory showed a preferential position in the anterior half of the volume of the nucleus, whereas chromosome 13 had a random position. This work shows a particular distribution of chromosome territories in the human sperm nucleus that could be related to mechanisms implicated in its specific functions. The analysis of more chromosomes and chromosomal structures, including the Y chromosome, would help to understand the structure of the human sperm chromatin, and its fundamental and clinical implications.
Cell Nucleus↗
Nonrandom distribution of metaphase AgNOR staining patterns on human acrocentric chromosomes.
The metaphase nucleolar organizer regions (NORs) contain ribosomal genes associated with proteins such as upstream binding factor (UBF) and RNA polymerase I (RPI). These genes are clustered in 10 loci of the human acrocentric chromosomes (13, 14, 15, 21, and 22). Some NOR-associated proteins, termed AgNOR proteins, can be specifically stained by silver. In this study we took advantage of technical advances in digital imaging, image restoration techniques, and factorial correspondence analysis (FCA) to study the different AgNOR staining patterns of metaphase chromosomes in human lymphocytes. Three predominant patterns could be distinguished: pair (47%), stick-like (28%), and unstained (18%) structures. By studying the frequency of occurrence of each pattern on different chromosomes, two groups could be defined. Chromosomes 13, 14, and 21 carried predominantly pair or stick-like AgNOR structures, whereas chromosomes 15 and 22 mainly carried pair AgNOR structures or remained unstained. We suggest that the different AgNOR shapes reflect both the number of ribosomal genes carried by each chromosome and the differential recruitment of active ribosomal genes in each NOR cluster. This is the first study showing a nonrandom distribution of AgNOR shape among acrocentric chromosomes.
Chromosomes, Human↗
Human XIST yeast artificial chromosome transgenes show partial X inactivation center function in mouse embryonic stem cells.
Initiation of X chromosome inactivation requires the presence, in cis, of the X inactivation center (XIC). The Xist gene, which lies within the XIC region in both human and mouse and has the unique property of being expressed only from the inactive X chromosome in female somatic cells, is known to be essential for X inactivation based on targeted deletions in the mouse. Although our understanding of the developmental regulation and function of the mouse Xist gene has progressed rapidly, less is known about its human homolog. To address this and to assess the cross-species conservation of X inactivation, a 480-kb yeast artificial chromosome containing the human XIST gene was introduced into mouse embryonic stem (ES) cells. The human XIST transcript was expressed and could coat the mouse autosome from which it was transcribed, indicating that the factors required for cis association are conserved in mouse ES cells. Cis inactivation as a result of human XIST expression was found in only a proportion of differentiated cells, suggesting that the events downstream of XIST RNA coating that culminate in stable inactivation may require species-specific factors. Human XIST RNA appears to coat mouse autosomes in ES cells before in vitro differentiation, in contrast to the behavior of the mouse Xist gene in undifferentiated ES cells, where an unstable transcript and no chromosome coating are found. This may not only reflect important species differences in Xist regulation but also provides evidence that factors implicated in Xist RNA chromosome coating may already be present in undifferentiated ES cells.
Animals↗
Quantitative assessment of the alteration of chromatin during the course of FISH procedures. Fluorescent in situ hybridization.
BACKGROUND: DNA denaturation, required for fluorescent in situ hybridization (FISH) experiments, is likely to induce chromatin alterations. Only few attempts have been made to quantify the extent of these perturbations. We propose a quality-control approach based on image analysis to monitor the effect of a procedure commonly used in FISH experiments. METHODS: Using DAPI as a probe, the same nuclei were successively imaged with a CCD camera after fixation, after permeabilization, and after thermal denaturation and hybridization with a centromeric probe. The modifications of the staining pattern were analyzed. Volumes of the FISH signals were measured using confocal imaging. RESULTS: DAPI staining combined with image analysis proved to be a sensitive tool to visualize the effects of different treatments used in FISH experiments. Permeabilization of nuclei after fixation has only limited impact on the chromatin. On the contrary, the denaturation procedure modifies the staining of DNA by DAPI, as well as the underlying chromatin structure as assessed by the increase of FISH signal volume with denaturation time. The protocol that involves a pre-fixation permeabilization step results in a more severe loss of chromatin structure. CONCLUSIONS: Our results clearly show that analysis of alterations of DAPI staining patterns is a useful monitoring tool to control and standardize hybridization procedures.
Amnion↗
Xist yeast artificial chromosome transgenes function as X-inactivation centers only in multicopy arrays and not as single copies.
X-chromosome inactivation in female mammals is controlled by the X-inactivation center (Xic). This locus is required for inactivation in cis and is thought to be involved in the counting process which ensures that only a single X chromosome remains active per diploid cell. The Xist gene maps to the Xic region and has been shown to be essential for inactivation in cis. Transgenesis represents a stringent test for defining the minimal region that can carry out the functions attributed to the Xic. Although YAC and cosmid Xist-containing transgenes have previously been reported to be capable of cis inactivation and counting, the transgenes were all present as multicopy arrays and it was unclear to what extent individual copies are functional. Using two different yeast artificial chromosomes (YACs), we have found that single-copy transgenes, unlike multicopy arrays, can induce neither inactivation in cis nor counting. These results demonstrate that despite their large size and the presence of Xist, the YACs that we have tested lack sequences critical for autonomous function with respect to X inactivation.
Animals↗
Assignment of the STOP gene (MAP6) to human chromosome bands 6p12-->p11 by fluorescence in situ hybridization.
Explore the source record for details and available documents.
Chromosome Banding↗
Optimization of nuclear transcript detection by FISH and combination with fluorescence immunocytochemical detection of transcription factors.
Detection of specific nuclear transcripts by fluorescence in situ hybridization (FISH) has constituted a major breakthrough in the study of the organization of transcription in the cell nucleus. Using the model of heat shock genes, we present an optimized procedure for nuclear transcripts that provides high efficiency for RNA detection and good preservation of cell morphology and nuclear texture. Using this procedure, we designed an original high-efficiency methodology combining FISH and fluorescence immunocytochemistry (FICC), which is used here for the simultaneous detection of heat-shock protein (hsp) nuclear transcripts and the specific heat-shock transcription factor 1 (HSF1). We show that the nuclear accumulation sites of HSF1 in heat-shocked cells do not correspond to the sites of transcription of the hsp70 gene.
Cell Nucleus↗
A mouse chromosome-specific YAC probe collection for in situ hybridization.
To facilitate the identification of mouse metaphase chromosomes by fluorescence in situ hybridization (FISH), a complete collection of mouse chromosome-specific markers has been established. Yeast artificial chromosome libraries were screened by polymerase chain reaction using primers for known loci. DNAs from positive clones were then tested by FISH. One probe per chromosome was selected on the basis of high specificity (nonchimerism) and strong fluorescence.
Animals↗
Transgenic mice carrying an Xist-containing YAC.
The initiation of X-chromosome inactivation in female mammals is controlled by a key locus, the X-inactivation centre (Xic). The Xist gene, which maps to the candidate region for Xic and is expressed exclusively from the inactive X chromosome, is thought to be an essential component of the Xic. To test whether sequences spanning several hundred kilobases and including Xist from the Xic region are capable of initiating inactivation, we have created a series of transgenic mice using a 460 kb yeast artificial chromosome (YAC). Analysis in these mice of the expression of Xist, of a LacZ reporter gene and of two genes in the region that are normally silent on the inactive X chromosome, suggests that essential sequences for Xist expression and X-inactivation may be absent in these transgenic animals.
Animals↗
Metaphase and interphase mapping by FISH: improvement of chromosome banding and signal resolution in interphase nuclei by means of iterative deconvolution.
FISH images obtained with conventional epifluorescence microscopes are always blurred by glare and out of focus light emissions. In order to restore high contrast images, a procedure based on a modelling of the optical system in the microscope was developed and used for the processing of images acquired with a cooled CCD camera mounted on a fluorescence microscope. This procedure was tested on images of both mouse and human chromosomes stained with DAP1 and on images of interphase nuclei hybridized with pairs of cosmid probes. This method improves the definition and the sharpness of the DAPI G-banding and thus facilitates and speeds up the identification of chromosomes. When performed on images of interphase cell nuclei, this procedure allows the discrimination of fluorescent signals which appear partially overlapping on raw images. This significant improvement of spatial resolution is of particular interest for ordering sets of probes on DNA fibers.
Animals↗
CK20 gene expression: technical limits for the detection of circulating tumor cells.
The suitability of CK20 mRNA expression as a marker for the detection of minimum residual disease in patients with cancer of epithelial origin was evaluated. A sensitive nested RT-PCR assay with multiple replicates was optimised to detect a minimum number of circulating tumor cells expressing cytokeratin 20 (CK20) mRNA. Using this optimal procedure, we examined CK20 mRNA expression in ten epithelial and seven leukemic cell lines, in eight bladder tumors, in peripheral blood samples from 18 tumor patients and from 29 healthy controls and in 8 bone marrow samples from healthy donors. CK20 mRNA was found in 13 of 18 (72%) blood samples from patients with cancer of epithelial origin and in all the epithelial tumor cells tested. However, CK20 mRNA was also detected in 21 of 29 (72%) bloods, in 8 of 8 bone marrow samples from healthy donors and in 4 of 7 leukemic cell lines. These results highlight a requirement for either determination of threshold levels of CK20 normal expression or the development of quantitative techniques to distinguish between a tumor-specific CK20 gene expression and a low level background transcription of this marker. These results would also advise caution in using CK20 as a tumor specific marker in clinical investigations.
Biomarkers, Tumor↗