Bar code screening on combed DNA for large rearrangements of the BRCA1 and BRCA2 genes in French breast cancer families.
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Adolescent↗
Biomedical subjects
A Bensimon
Publications and source records attributed to A Bensimon.
Targeting the molecular mechanism of DNA replication.
Genome stability is crucial for the complete maintenance of the cellular pathways that govern the cell cycle. As a result of irregularities in DNA replication occurring throughout the S phase, key genes that regulate cell cycle pathways are damaged, giving rise to single-base mutations and chromosomal aberrations. Thus, the efficient replication of the genome, which depends on a precise temporal and spatial pattern of activation of origins of replication, is greatly impaired. The approach discussed below aims at monitoring the replication pattern and the kinetics of replication throughout the entire genome of living cells. It could shed light on the mechanisms by which drugs act on DNA replication and, moreover, it might assist the discovery and design of novel drugs that inhibit cell proliferation under pathophysiological conditions.
Journal Article↗
Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains.
Eukaryotic chromosome replication is initiated from numerous origins and its activation is temporally controlled by cell cycle and checkpoint mechanisms. Yeast has been very useful in defining the genetic elements required for initiation of DNA replication, but simple and precise tools to monitor S phase progression are lacking in this model organism. Here we describe a TK(+) yeast strain and conditions that allow incorporation of exogenous BrdU into genomic DNA, along with protocols to detect the sites of DNA synthesis in yeast nuclei or on combed DNA molecules. S phase progression is monitored by quantification of BrdU in total yeast DNA or on individual chromosomes. Using these tools we show that yeast chromosomes replicate synchronously and that DNA synthesis occurs at discrete subnuclear foci. Analysis of BrdU signals along single DNA molecules from hydroxyurea-arrested cells reveals that replication forks stall 8-9 kb from origins that are placed 46 kb apart on average. Quantification of total BrdU incorporation suggests that 190 'early' origins have fired in these cells and that late replicating territories might represent up to 40% of the yeast genome. More generally, the methods outlined here will help understand the kinetics of DNA replication in wild-type yeast and refine the phenotypes of several mutants.
Blotting, Southern↗
Color bar coding the BRCA1 gene on combed DNA: a useful strategy for detecting large gene rearrangements.
Genetic linkage data have shown that alterations of the BRCA1 gene are responsible for the majority of hereditary breast and ovarian cancers. BRCA1 germline mutations, however, are found less frequently than expected. Mutation detection strategies, which are generally based on the polymerase chain reaction, therefore focus on point and small gene alterations. These approaches do not allow for the detection of large gene rearrangements, which also can be involved in BRCA1 alterations. Indeed, a few of them, spread over the entire BRCA1 gene, have been detected recently by Southern blotting or transcript analysis. We have developed an alternative strategy allowing a panoramic view of the BRCA1 gene, based on dynamic molecular combing and the design of a full four-color bar code of the BRCA1 region. The strategy was tested with the study of four large BRCA1 rearrangements previously reported. In addition, when screening a series of 10 breast and ovarian cancer families negatively tested for point mutation in BRCA1/2, we found an unreported 17-kb BRCA1 duplication encompassing exons 3 to 8. The detection of rearrangements as small as 2 to 6 kb with respect to the normal size of the studied fragment is achieved when the BRCA1 region is divided into 10 fragments. In addition, as the BRCA1 bar code is a morphologic approach, the direct observation of complex and likely underreported rearrangements, such as inversions and insertions, becomes possible.
Breast Neoplasms↗
Identification of a large rearrangement of the BRCA1 gene using colour bar code on combed DNA in an American breast/ovarian cancer family previously studied by direct sequencing.
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Adult↗
Improvement of FISH mapping resolution on combed DNA molecules by iterative constrained deconvolution: a quantitative study.
Image restoration approaches, such as digital deconvolution, are becoming widely used for improving the quality of microscopic images. However, no quantification of the gain in resolution of fluorescence images is available. We show that, after iterative constrained deconvolution, fluorescent cosmid signals appear to be 25% smaller, and 1.2-kb fragment signals on combed molecules faithfully display the expected length.
Chromosomes, Artificial, Yeast↗
Replication fork density increases during DNA synthesis in X. laevis egg extracts.
Duplication of the eukaryotic genome depends on the temporal and spatial organization of DNA replication during the cell cycle. To investigate the genomic organization of DNA replication in a higher eukaryote, multiple origins of replication must be simultaneously analyzed over large regions of the genome as DNA synthesis progresses through S phase of the cell cycle. We have employed a novel technique that allows for the quantitative analysis of DNA replication on a genome wide basis. The technique involves stretching and aligning individual DNA molecules on a glass surface. As a model system, Xenopus laevis egg extract was used to differentially label sperm chromatin at successive time points after the start of DNA synthesis. The differentially labeled DNA allows earlier and later replicating sequences to be distinguished, and hence the sites of DNA synthesis at any given time can be directly visualized. Genomic DNA was extracted, and measurements made on the linearized molecules provided a comprehensive analysis of the spatial and temporal organization of DNA replication in the X. laevis in vitro replication system. It was found that: (i) DNA synthesis initiates asynchronously at irregular intervals but continuously as DNA replication advances; and (ii) that the frequency of initiation (the number of activated origins per kilobase) increases as DNA synthesis nears completion. The implications of these findings for the regulation of DNA replication in early embryos is discussed.
Animals↗
Encephalitozoon cuniculi (Microspora) genome: physical map and evidence for telomere-associated rDNA units on all chromosomes.
A restriction map of the 2.8-Mb genome of the unicellular eukaryote Encephalitozoon cuniculi (phylum Microspora), a mammal-infecting intracellular parasite, has been constructed using two restriction enzymes with 6 bp recognition sites (Bss HII and Mlu I). The fragments resulting from either single digestions of the whole molecular karyotype or double digestions of 11 individual chromosomes have been separated by two-dimensional pulsed field gel electrophoresis (2D-PFGE) procedures. The average distance between successive restriction sites is approximately 19 kb. The terminal regions of the chromosomes show a common pattern covering approximately 15 kb and including one 16S-23S rDNA unit. Results of hybridisation and molecular combing experiments indicate a palindromic-like orientation of the two subtelomeric rDNA copies on each chromosome. We have also located 67 DNA markers (clones from a partial E. cuniculi genomic library) by hybridisation to restriction fragments. Partial or complete sequencing has revealed homologies with known protein-coding genes for 32 of these clones. Evidence for two homologous chromosomes III, with a size difference (3 kb) related to a subtelomeric deletion/insertion event, argues for diploidy of E.cuniculi. The physical map should be useful for both the whole genome sequencing project and studies on genome plasticity of this widespread parasite.
Animals↗
Quantifying single gene copy number by measuring fluorescent probe lengths on combed genomic DNA.
An approach was developed for the quantification of subtle gains and losses of genomic DNA. The approach relies on a process called molecular combing. Molecular combing consists of the extension and alignment of purified molecules of genomic DNA on a glass coverslip. It has the advantage that a large number of genomes can be combed per coverslip, which allows for a statistically adequate number of measurements to be made on the combed DNA. Consequently, a high-resolution approach to mapping and quantifying genomic alterations is possible. The approach consists of applying fluorescence hybridization to the combed DNA by using probes to identify the amplified region. Measurements then are made on the linear hybridization signals to ascertain the region's exact size. The reliability of the approach first was tested for low copy number amplifications by determining the copy number of chromosome 21 in a normal and trisomy 21 cell line. It then was tested for high copy number amplifications by quantifying the copy number of an oncogene amplified in the tumor cell line GTL-16. These results demonstrate that a wide range of amplifications can be accurately and reliably quantified. The sensitivity and resolution of the approach likewise was assessed by determining the copy number of a single allele (160 kb) alteration.
Bacteriophage lambda↗
Physical mapping of the CC-chemokine gene cluster on the human 17q11. 2 region.
Chemokines are a family of small secreted proteins that are involved in the trafficking of leukocytes by acting on G-protein-coupled receptors. Specific chemokines are also implicated in the regulation of angiogenesis and mobilization of hematopoietic cell precursors. Chemokines are subdivided into four groups on the basis of the relative positions of their conserved cysteines. For the CC-chemokine group, in which the first two (of four) conserved cysteines are adjacent, 22 members have been described so far. In this work, we have analyzed the genomic organization of these genes. We first assigned the genes encoding CC-chemokines to chromosomal regions and organized their relative positioning by using two radiation hybrid panels. Fifteen CC-chemokine genes were shown to be clustered within the 17q11.2 region of the human genome. These genes appeared to be segregated into two subclusters separated by about 2. 25 Mb (9 cR). Contigs of bacterial artificial chromosomes (BAC) covering these two subclusters were subsequently isolated and the localizations of the CC-chemokine genes within these contigs determined. The relative positioning of the BAC clones was determined with the help of fluorescence hybridization on combed genomic DNA. The cluster organization of the various CC-chemokine genes in the genome was found to be grossly consistent with their structural similarities. This map of the CC-chemokine gene cluster should facilitate the determination of the full sequence of the chromosomal region.
Chemokines, CC↗
Imaging of single DNA molecule: applications to high-resolution genomic studies.
Single molecule analysis of DNA has revealed new insights into its structural and physical properties. The application of new methods for manipulating and visualizing DNA has resulted in important advances in high-resolution physical mapping of the genome and quantitative cytogenetic studies of genomic abnormalities (Lichter 1997). Studies of single molecules of DNA have employed a variety of approaches including electron microscopy, atomic force microscopy, scanning-tunneling microscopy and fluorescence microscopy. A number of new technologies have recently been developed to exploit fluorescence microscopy's full potential for genomic analysis and the fine mapping of subtle genetic alterations. In the case of the latter application, particular emphasis has been placed on developing new methods for stretching DNA for high-resolution fluorescence in-situ hybridization studies. We have recently described a process called molecular combing according to which single DNA molecules bound by their extremities to a solid surface are uniformly stretched and aligned by a receding air/water interface (Bensimon et al. 1994). In the following, we will review recent developments concerning molecular combing and discuss its current and potential applications for the high-resolution mapping of the human genome, the detection and quantification of subtle genomic imbalances and the positional cloning of disease-related genes.
Chromosome Aberrations↗
Mapping of the CCXCR1, CX3CR1, CCBP2 and CCR9 genes to the CCR cluster within the 3p21.3 region of the human genome.
Human CC-chemokine receptor genes are known to be clustered. The detailed structure of this cluster was established by radiation hybrid mapping, and organization of BAC contigs by fluorescence hybridization on combed genomic DNA. A main cluster of six genes (CCR1, CCR3, CCRL2, CCR5, CCR2 and CCXCR1), covered by four BACs, was mapped to the 3p21.3 region of the human genome. Five other genes (CCR9, CCBP2, CX3CR1, CCR8 and CCR4) were found to be spread over a relatively large region between this main cluster and the 3p telomere.
CX3C Chemokine Receptor 1↗
High-resolution mapping of the X-linked lymphoproliferative syndrome region by FISH on combed DNA.
X-linked lymphoproliferative syndrome is an inherited immunodeficiency for which the responsible gene is currently unknown. Several megabase-sized deleted regions mapping to Xq25 have been identified in XLP patients, and more recently a 130-kb deletion has been reported (Lamartine et al., 1996; Lanyi et al., 1996). To establish a physical map of this deleted region and to identify the XLP gene, two cosmid contigs were established (Lamartine et al., 1996). However, the physical map of this region is still uncompleted and controversial and three points remain unsolved: (1) the centromeric-telomeric orientation of the whole region, (2) the relative orientation of the two contigs, and (3) the size of the gap between the two contigs. To provide a definitive answer to these questions, high-resolution mapping by fluorescence in situ hybridization on combed DNA and molecular approaches were combined to establish the physical map of the XLP region over 600 kb. Our results identified a gap of 150 kb between the two contigs, established the relative orientation of one contig to the other, and determine the centromeric-telomeric orientation of the whole region. Our results show that the order of the marker over this region is: cen.1D10T7-DF83-DXS982.tel.
Chromosome Mapping↗
Dynamic molecular combing: stretching the whole human genome for high-resolution studies.
DNA in amounts representative of hundreds of eukaryotic genomes was extended on silanized surfaces by dynamic molecular combing. The precise measurement of hybridized DNA probes was achieved directly without requiring normalization. This approach was validated with the high-resolution mapping of cosmid contigs on a yeast artificial chromosome (YAC) within yeast genomic DNA. It was extended to human genomic DNA for precise measurements ranging from 7 to 150 kilobases, of gaps within a contig, and of microdeletions in the tuberous sclerosis 2 gene on patients' DNA. The simplicity, reproducibility, and precision of this approach makes it a powerful tool for a variety of genomic studies.
Calpain↗
High-resolution comparative hybridization to combed DNA fibers.
Comparative genomic hybridization (CGH) has proven to be a comprehensive new tool to detect genetic imbalances in genomic DNA. However, the resolution of this method carried out on normal human metaphase spreads is limited to low copy number gains and losses of > or = 10 Mb. An improved resolution allowing the detection of copy number representations of single genes would strongly enhance the applicability of CGH as a diagnostic and research tool. This goal may be achieved when metaphase chromosomes are replaced by an array of target DNAs representing the genes of interest. To explore the feasibility of such a development in a model system we used cosmid MA2B3, which encompasses about 35 kb in the vicinity of exon 48 of the human dystrophin gene. Linearized cosmid fibers were attached to a glass surface and aligned in parallel by "molecular combing". Two-color fluorescence in situ suppression hybridization was performed on these cosmid fibers with probe mixtures containing different ratios (ranging from 1:2 to 4:1) of biotin- and digoxigenin-labeled MA2B3 cosmid DNAs. For each mixture fluorescence ratios were determined for 40-50 individual combed DNA molecules. In two series comprising a total of 651 molecules the median fluorescence ratio measurements revealed a linear relationship with the chosen probe ratios. Our study demonstrates that fluorescence ratio measurements on single DNA molecules can be performed successfully.
Biotin↗
pH-dependent specific binding and combing of DNA.
Recent developments in the rapid sequencing, mapping, and analysis of DNA rely on the specific binding of DNA to specially treated surfaces. We show here that specific binding of DNA via its unmodified extremities can be achieved on a great variety of surfaces by a judicious choice of the pH. On hydrophobic surfaces the best binding efficiency is reached at a pH of approximately 5.5. At that pH a approximately 40-kbp DNA is 10 times more likely to bind by an extremity than by a midsegment. A model is proposed to account for the differential adsorption of the molecule extremities and midsection as a function of pH. The pH-dependent specific binding can be used to align anchored DNA molecules by a receding meniscus, a process called molecular combing. The resulting properties of the combed molecules will be discussed.
Adsorption↗
The elasticity of a single supercoiled DNA molecule.
Single linear DNA molecules were bound at multiple sites at one extremity to a treated glass cover slip and at the other to a magnetic bead. The DNA was therefore torsionally constrained. A magnetic field was used to rotate the beads and thus to coil and pull the DNA. The stretching force was determined by analysis of the Brownian fluctuations of the bead. Here the elastic behavior of individual lambda DNA molecules over- and underwound by up to 500 turns was studied. A sharp transition was discovered from a low to a high extension state at a force of approximately 0.45 piconewtons for underwound molecules and at a force of approximately 3 piconewtons for overwound ones. These transitions, probably reflecting the formation of alternative structures in stretched coiled DNA molecules, might be relevant for DNA transcription and replication.
Bacteriophage lambda↗
Stretching DNA with a receding meniscus: Experiments and models.
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Journal Article↗