Walking, cloning, and mapping with yeast artificial chromosomes: a contig encompassing D21S13 and D21S16.

Chromosome 21 has often been used as a model system for the development of genome mapping and cloning strategies in humans. In this report methods for systematic chromosome walking, cloning, and mapping are exemplified in the construction of a 1.5-Mb yeast artificial chromosome (YAC) contig encompassing and extending 400 kb beyond each of the genetic loci D21S13 and D21S16. Isolation of insert-terminal sequences from YACs in this contig provides a set of closely spaced physical markers. These have been used to generate a long-range genomic restriction map.     

Single-molecule analysis of genome rearrangements in cancer

Rearrangements of the genome can be detected by microarray methods and massively parallel sequencing, which identify copy-number alterations and breakpoint junctions, but these techniques are poorly suited to reconstructing the long-range organization of rearranged chromosomes, for example, to distinguish between translocations and insertions. The single-DNA-molecule technique HAPPY mapping is a method for mapping normal genomes that should be able to analyse genome rearrangements, i.e. deviations from a known genome map, to assemble rearrangements into a long-range map. We applied HAPPY mapping to cancer cell lines to show that it could identify rearrangement of genomic segments, even in the presence of normal copies of the genome. We could distinguish a simple interstitial deletion from a copy-number loss at an inversion junction, and detect a known translocation. We could determine whether junctions detected by sequencing were on the same chromosome, by measuring their linkage to each other, and hence map the rearrangement. Finally, we mapped an uncharacterized reciprocal translocation in the T-47D breast cancer cell line to about 2 kb and hence cloned the translocation junctions. We conclude that HAPPY mapping is a versatile tool for determining the structure of rearrangements in the human genome.     

Optical PCR: Genomic analysis by long-range PCR and optical mapping

Optical mapping is an approach for the rapid, automated, non-electrophoretic construction of ordered restriction maps of DNA from ensembles of single molecules. Previously, we used optical mapping to make high-resolution maps of large insert clones such as bacterial artificial chromosomes (BAC) and large genomic DNA molecules. Here, we describe a combination of optical mapping and long-range polymerase chain reaction (PCR), in a process we term optical PCR, which enables automated construction of ordered restriction maps of long-range PCR products spanning human genomic loci. Specifically, we amplified three long PCR products, each averaging 14.6 kb in length, which span the 37-kb human tissue plasminogen activator (TPA) gene. PCR products were surface mounted in gridded arrays, and samples were mapped in parallel with either ScaI, XmnI, HpaI, ClaI, or BglII. A contig of overlapping high-resolution maps was generated, which agreed closely with maps predicted from sequence data. The data demonstrate an approach to construct physical maps of genomic loci where very little prior sequence information exists, since the only sequence needed is that required to anchor PCR primers. Large segments of genomic DNA (within the practical limits imposed by long-range PCR) can be mapped quickly and to high resolution without the use of cloning vectors. Received: 9 February 1999 / Accepted: 26 May 1999     

Exon Mapping by Fiber-FISH or LR-PCR

In this study we systematically assessed the sensitivity limits of fiber-FISH in model experiments. Exonic fragments and cDNAs with exon sizes of ≥200 bp could be mapped on their cognate cosmid. This positional fiber-FISH mapping was validated by long-range PCR. It is expected that these two independent mapping approaches will help to refine current available gene maps and show their applicability in fine mapping of sequence-tagged sites or expressed sequence tags. Also, they will be useful in resolving gene structures by mapping exon and intron locations.     

Monte-Carlo simulations of strongly interacting dipolar fluids between two conducting walls

We report Monte-Carlo simulation results for a strongly interacting dipolar soft sphere (DSS) fluid confined between two conducting, planar walls. The long-range dipolar interactions, including contributions from the “image dipoles” in the metal, are handled by mapping onto a problem with three-dimensional (3d) periodicity which can be treated by conventional Ewald summation methods. Considering two different wall separations our results indicate the occurence of wall-induced local and long-range ordering very similar to the related case of insulating walls. To understand this behavior we present ground-state (lattice) calculations on the basis of the Ewald sums, as well as simple macroscopic arguments appropriate for dipolar systems between conducting walls.     

A model for nucleotide sequences.

We propose a model for generating "artificial" nucleotide sequences and, by the method of mapping those sequences onto a "DNA-walk," we analyze the presence of correlation between nucleotides. Artificial sequences are constructed considering, basically, interactions between first neighbors and between more distant units. We show that long-range correlations may be favored by the occurrence of intrastrand interactions, which give a nonlinear characteristic to the sequence.     

人Xp11.2区4.3MbYAC重叠群：大尺度限制图与CpG岛分析

人Ｘｐ１１．２区域具有重要的医学遗传学和基础遗传学价值,它包含很多遗传疾病基因,且至少包含一个逃避Ｘ染色体失活的位点,非常规的基化多态也有发现。我们利用这一区域已知的一系列ＤＮＡ位标,从我们构建的ＹＡＣ库中筛选出一系列ＹＡＣ克隆。     

Physical maps of 4p16.3, the area expected to contain the Huntington disease mutation

The gene for Huntington disease, a neurodegenerative disorder with autosomal dominant inheritance, has been localized to the terminal portion of the short arm of human chromosome 4 (4p16.3) by linkage analysis. Since eventual isolation of the gene requires the application of high-resolution genetic analysis coupled with long-range DNA mapping and cloning techniques, we have constructed a physical map of the chromosomal region 4p16.3 using more than 20 independently derived probes. We have grouped these markers into three clusters which have been ordered and oriented by genetic and somatic cell genetic mapping information. The mapped region extends from D4S10 (G8) toward the telomere and covers minimally 5 Mb.     

Cloning high molecular weight DNA fragments by the bacteriophage P1 system.

The cloning of high molecular weight genomic DNA promises to provide the means of mapping chromosomes, isolating genes, and understanding long-range effects on gene expression. This review describes the background and some recent advances in cloning of high molecular weight DNA using the bacteriophage P1 system.     

Super-resolution optical DNA Mapping via DNA methyltransferase-directed click chemistry

We demonstrate an approach to optical DNA mapping, which enables near single-molecule characterization of whole bacteriophage genomes. Our approach uses a DNA methyltransferase enzyme to target labelling to specific sites and copper-catalysed azide-alkyne cycloaddition to couple a fluorophore to the DNA. We achieve a labelling efficiency of ∼70% with an average labelling density approaching one site every 500 bp. Such labelling density bridges the gap between the output of a typical DNA sequencing experiment and the long-range information derived from traditional optical DNA mapping. We lay the foundations for a wider-scale adoption of DNA mapping by screening 11 methyltransferases for their ability to direct sequence-specific DNA transalkylation; the first step of the DNA labelling process and by optimizing reaction conditions for fluorophore coupling via a click reaction. Three of 11 enzymes transalkylate DNA with the cofactor we tested (a readily prepared s-adenosyl-l-methionine analogue).     

Yeast artificial chromosome-based genome mapping: some lessons from Xq24-q28.

Yeast artificial chromosomes (YACs) have recently provided a potential route to long-range coverage of complex genomes in contiguous cloned DNA. In a pilot project for 50 Mb (1.5% of the human genome), a variety of techniques have been applied to assemble Xq24-q28 YAC contigs up to 8 Mb in length and assess their quality. The results indicate the relative strength of several approaches and support the adequacy of YAC-based methods for mapping the human genome.     

Physical mapping of 60 DNA markers in the p21.1----q21.3 region of the human X chromosome.

Using a panel of human/rodent somatic cell hybrids and human lymphoblast lines segregating 18 different human X-chromosome rearrangements and deletions, we have assigned 60 DNA markers to the physical map of the X chromosome from Xp21.1 to Xq21.3. Data from Southern blot hybridization and polymerase chain reaction (PCR) amplification assign these markers to 15 primary map intervals. This provides a basis for further long-range cloning and mapping of the pericentromeric region of the X chromosome.     

Nanoscale Cell Wall Deformation Impacts Long-Range Bacterial Adhesion Forces on Surfaces

Adhesion of bacteria occurs on virtually all natural and synthetic surfaces and is crucial for their survival. Once they are adhering, bacteria start growing and form a biofilm, in which they are protected against environmental attacks. Bacterial adhesion to surfaces is mediated by a combination of different short- and long-range forces. Here we present a new atomic force microscopy (AFM)-based method to derive long-range bacterial adhesion forces from the dependence of bacterial adhesion forces on the loading force, as applied during the use of AFM. The long-range adhesion forces of wild-type Staphylococcus aureus parent strains (0.5 and 0.8 nN) amounted to only one-third of these forces measured for their more deformable isogenic Δpbp4 mutants that were deficient in peptidoglycan cross-linking. The measured long-range Lifshitz-Van der Waals adhesion forces matched those calculated from published Hamaker constants, provided that a 40% ellipsoidal deformation of the bacterial cell wall was assumed for the Δpbp4 mutants. Direct imaging of adhering staphylococci using the AFM peak force-quantitative nanomechanical property mapping imaging mode confirmed a height reduction due to deformation in the Δpbp4 mutants of 100 to 200 nm. Across naturally occurring bacterial strains, long-range forces do not vary to the extent observed here for the Δpbp4 mutants. Importantly, however, extrapolating from the results of this study, it can be concluded that long-range bacterial adhesion forces are determined not only by the composition and structure of the bacterial cell surface but also by a hitherto neglected, small deformation of the bacterial cell wall, facilitating an increase in contact area and, therewith, in adhesion force.     

Unusually high recombination rate detected in the sex locus region of the honey bee (Apis mellifera)

Sex determination in Hymenoptera is controlled by haplo-diploidy in which unfertilized eggs develop into fertile haploid males. A single sex determination locus with several complementary alleles was proposed for Hymenoptera [so-called complementary sex determination (CSD)]. Heterozygotes at the sex determination locus are normal, fertile females, whereas diploid zygotes that are homozygous develop into sterile males. This results in a strong heterozygote advantage, and the sex locus exhibits extreme polymorphism maintained by overdominant selection. We characterized the sex-determining region by genetic linkage and physical mapping analyses. Detailed linkage and physical mapping studies showed that the recombination rate is <44 kb/cM in the sex-determining region. Comparing genetic map distance along the linkage group III in three crosses revealed a large marker gap in the sex-determining region, suggesting that the recombination rate is high. We suggest that a "hotspot" for recombination has resulted here because of selection for combining favorable genotypes, and perhaps as a result of selection against deleterious mutations. The mapping data, based on long-range restriction mapping, suggest that the Q DNA-marker is within 20,000 bp of the sex locus, which should accelerate molecular analyses.     

Yeast artificial chromosome-based genome mapping: Some lessons from Xq24–q28

Yeast artificial chromosomes (YACs) have recently provided a potential route to long-range coverage of complex genomes in contiguous cloned DNA. In a pilot project for 50 Mb (1.5% of the human genome), a variety of techniques have been applied to assemble Xq24–q28 YAC contigs up to 8 Mb in length and assess their quality. The results indicate the relative strength of several approaches and support the adequacy of YAC-based methods for mapping the human genome.     

Constructing plant radiation hybrid panels

Radiation hybrid (RH) mapping, a somatic cell genetic technique, has been developed in animal systems as a general approach for the construction of long-range physical maps of chromosomes. This statistical method relies on X-ray induced breakage of chromosomes to determine the physical distance between markers, as well as their order on the chromosome. The method can be applied to single chromosomes or across the whole genome. The generation of plant (barley) radiation hybrids and their culture in vitro is described here. PCR-based marker systems are used to verify hybrid status and to demonstrate genome coverage. RH panels of the type generated can be used for physical mapping, map-based cloning, or sequence contig assembly. RH resources will greatly aid the physical characterisation of crop plants with large genomes.     

Intracranial EEG and human brain mapping

This review is an attempt to highlight the value of human intracranial recordings (intracranial electro-encephalography, iEEG) for human brain mapping, based on their technical characteristics and based on the corpus of results they have already yielded. The advantages and limitations of iEEG recordings are introduced in detail, with an estimation of their spatial and temporal resolution for both monopolar and bipolar recordings. The contribution of iEEG studies to the general field of human brain mapping is discussed through a review of the effects observed in the iEEG while patients perform cognitive tasks. Those effects range from the generation of well-localized evoked potentials to the formation of large-scale interactions between distributed brain structures, via long-range synchrony in particular. A framework is introduced to organize those iEEG studies according to the level of complexity of the spatio-temporal patterns of neural activity found to correlate with cognition. This review emphasizes the value of iEEG for the study of large-scale interactions, and describes in detail the few studies that have already addressed this point.