Biomedical subjects

Marie-Laure Yaspo

Publications and source records attributed to Marie-Laure Yaspo.

8 recordsLinked to original sources

PubMed2006

An efficient and economic enhancer mix for PCR.

Polymerase chain reaction (PCR) has become a fundamental technique in molecular biology. Nonetheless, further improvements of the existing protocols are required to broaden the applicability of PCR for routine diagnostic purposes, to enhance the specificity and the yield of PCRs as well as to reduce the costs for high-throughput applications. One known problem typically reported in PCR experiments is the poor amplification of GC-rich DNA sequences. Here we designed and tested a novel effective and low-cost PCR enhancer, a concentration-dependent combination of betaine, dithiothreitol, and dimethyl sulfoxide that broadly enhanced the quantitative and/or qualitative output of PCRs. Additionally, we showed that the performances of this enhancer mix are comparable to those of commercially available PCR additives and highly effective with different DNA polymerases. Thus, we propose the routine application of this PCR enhancer mix for low- and high-throughput experiments.

Enhancer Elements, Genetic↗

PubMed2006

Cell array-based intracellular localization screening reveals novel functional features of human chromosome 21 proteins.

BACKGROUND: Trisomy of human chromosome 21 (Chr21) results in Down's syndrome, a complex developmental and neurodegenerative disease. Molecular analysis of Down's syndrome, however, poses a particular challenge, because the aneuploid region of Chr21 contains many genes of unknown function. Subcellular localization of human Chr21 proteins may contribute to further understanding of the functions and regulatory mechanisms of the genes that code for these proteins. Following this idea, we used a transfected-cell array technique to perform a rapid and cost-effective analysis of the intracellular distribution of Chr 21 proteins. RESULTS: We chose 89 genes that were distributed over the majority of 21q, ranging from RBM11 (14.5 Mb) to MCM3AP (46.6 Mb), with part of them expressed aberrantly in the Down's syndrome mouse model. Open reading frames of these genes were cloned into a mammalian expression vector with an amino-terminal His6 tag. All of the constructs were arrayed on glass slides and reverse transfected into HEK293T cells for protein expression. Co-localization detection using a set of organelle markers was carried out for each Chr21 protein. Here, we report the subcellular localization properties of 52 proteins. For 34 of these proteins, their localization is described for the first time. Furthermore, the alteration in cell morphology and growth as a result of protein over-expression for claudin-8 and claudin-14 genes has been characterized. CONCLUSION: The cell array-based protein expression and detection approach is a cost-effective platform for large-scale functional analyses, including protein subcellular localization and cell phenotype screening. The results from this study reveal novel functional features of human Chr21 proteins, which should contribute to further understanding of the molecular pathology of Down's syndrome.

Cell Cycle↗

PubMed2005

Primary differentiation in the human blastocyst: comparative molecular portraits of inner cell mass and trophectoderm cells.

The primary differentiation event during mammalian development occurs at the blastocyst stage and leads to the delineation of the inner cell mass (ICM) and the trophectoderm (TE). We provide the first global mRNA expression data from immunosurgically dissected ICM cells, TE cells, and intact human blastocysts. Using a cDNA microarray composed of 15,529 cDNAs from known and novel genes, we identify marker transcripts specific to the ICM (e.g., OCT4/POU5F1, NANOG, HMGB1, and DPPA5) and TE (e.g., CDX2, ATP1B3, SFN, and IPL), in addition to novel ICM- and TE-specific expressed sequence tags. The expression patterns suggest that the emergence of pluripotent ICM and TE cell lineages from the morula is controlled by metabolic and signaling pathways, which include inter alia, WNT, mitogen-activated protein kinase, transforming growth factor-beta, NOTCH, integrin-mediated cell adhesion, phosphatidylinositol 3-kinase, and apoptosis. These data enhance our understanding of the first step in human cellular differentiation and, hence, the derivation of both embryonic stem cells and trophoblastic stem cells from these lineages.

Apoptosis↗

PubMed2004

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division.

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.

Cell Division↗

PubMed2004

Regulation of the MID1 protein function is fine-tuned by a complex pattern of alternative splicing.

Clinical features of Opitz BBB/G syndrome are confined to defects of the developing ventral midline, whereas the causative gene, MID1, is ubiquitously expressed. Therefore, a non-redundant physiological function of the MID1 product appears to be developmentally restricted. Here, we report the identification of several alternative MID1 exons in human, mouse and fugu. We show that splice variants of the MID1 gene that are comparable in terms of function occur in the three organisms, suggesting an important role in the regulation of the MID1 protein function. Accordingly, we observed differential MID1 transcript patterns in a tissue-specific manner by Northern blot and RT-PCR. The identified splice variants cause loss-of-function effects via several mechanisms. Some introduce a stop codon followed by a novel poly(A(+)) tail, leading to the formation of C-terminally truncated proteins. Dominant negative effects through altered binding to the MID1-interacting protein alpha4 in vitro could be demonstrated in a couple of cases. Others carry premature termination codons without poly(A(+)) tails. These are degraded by nonsense mediated mRNA decay (NMD). Our data reveal a mechanism conserved in human, mouse and fugu that regulates developmentally restricted MID1 activity and suggest NMD to be critical in the translational regulation of a ubiquitously transcribed mRNA.

Alternative Splicing↗

PubMed2004

Transcript level alterations reflect gene dosage effects across multiple tissues in a mouse model of down syndrome.

Human trisomy 21, which results in Down syndrome (DS), is one of the most complicated congenital genetic anomalies compatible with life, yet little is known about the molecular basis of DS. It is generally accepted that chromosome 21 (Chr21) transcripts are overexpressed by about 50% in cells with an extra copy of this chromosome. However, this assumption is difficult to test in humans due to limited access to tissues, and direct support for this idea is available for only a few Chr21 genes or in a limited number of tissues. The Ts65Dn mouse is widely used as a model for studies of DS because it is at dosage imbalance for the orthologs of about half of the 284 Chr21 genes. Ts65Dn mice have several features that directly parallel developmental anomalies of DS. Here we compared the expression of 136 mouse orthologs of Chr21 genes in nine tissues of the trisomic and euploid mice. Nearly all of the 77 genes which are at dosage imbalance in Ts65Dn showed increased transcript levels in the tested tissues, providing direct support for a simple model of increased transcription proportional to the gene copy number. However, several genes escaped this rule, suggesting that they may be controlled by additional tissue-specific regulatory mechanisms revealed in the trisomic situation.

Animals↗

PubMed2002

A gene expression map of human chromosome 21 orthologues in the mouse.

The DNA sequence of human chromosome 21 (HSA21) has opened the route for a systematic molecular characterization of all of its genes. Trisomy 21 is associated with Down's syndrome, the most common genetic cause of mental retardation in humans. The phenotype includes various organ dysmorphies, stereotypic craniofacial anomalies and brain malformations. Molecular analysis of congenital aneuploidies poses a particular challenge because the aneuploid region contains many protein-coding genes whose function is unknown. One essential step towards understanding their function is to analyse mRNA expression patterns at key stages of organism development. Seminal works in flies, frogs and mice showed that genes whose expression is restricted spatially and/or temporally are often linked with specific ontogenic processes. Here we describe expression profiles of mouse orthologues to HSA21 genes by a combination of large-scale mRNA in situ hybridization at critical stages of embryonic and brain development and in silico (computed) mining of expressed sequence tags. This chromosome-scale expression annotation associates many of the genes tested with a potential biological role and suggests candidates for the pathogenesis of Down's syndrome.

Animals↗

PubMed2002

Construction and analysis of a human-chimpanzee comparative clone map.

The recently released human genome sequences provide us with reference data to conduct comparative genomic research on primates, which will be important to understand what genetic information makes us human. Here we present a first-generation human-chimpanzee comparative genome map and its initial analysis. The map was constructed through paired alignment of 77,461 chimpanzee bacterial artificial chromosome end sequences with publicly available human genome sequences. We detected candidate positions, including two clusters on human chromosome 21 that suggest large, nonrandom regions of difference between the two genomes.

Animals↗