Host response to EBV infection in X-linked lymphoproliferative disease results from mutations in an SH2-domain encoding gene.

X-linked lymphoproliferative syndrome (XLP or Duncan disease) is characterized by extreme sensitivity to Epstein-Barr virus (EBV), resulting in a complex phenotype manifested by severe or fatal infectious mononucleosis, acquired hypogammaglobulinemia and malignant lymphoma. We have identified a gene, SH2D1A, that is mutated in XLP patients and encodes a novel protein composed of a single SH2 domain. SH2D1A is expressed in many tissues involved in the immune system. The identification of SH2D1A will allow the determination of its mechanism of action as a possible regulator of the EBV-induced immune response.

The identification of SNPs with indeterminate positions using the Equine SNP50 BeadChip.

We have used linkage disequilibrium (LD) to identify single nucleotide polymorphisms (SNPs) on the Illumina Equine SNP50 BeadChip, which may be incorrectly positioned on the genome map. A total of 1201 Thoroughbred horses were genotyped using the Illumina Equine SNP50 BeadChip. LD was evaluated in a pairwise fashion between all autosomal SNPs, both within and across chromosomes. Filters were then applied to the data, firstly to identify SNPs that may have been mapped to the wrong chromosome and secondly to identify SNPs that may have been incorrectly positioned within chromosomes. We identified a single SNP on ECA28, which showed low LD with neighbouring SNPs but considerable LD with a group of SNPs on ECA10. Furthermore, a cluster of SNPs on ECA5 showed unusually low LD with surrounding SNPs. A total of 39 SNPs met the criteria for unusual within-chromosome LD. The results of this study indicate that some SNPs may be misplaced. This finding is significant, as misplaced SNPs may lead to difficulties in the application of genomic methods, such as homozygosity mapping, for which SNP order is important.

Linkage disequilibrium and historical effective population size in the Thoroughbred horse.

Many genomic methodologies rely on the presence and extent of linkage disequilibrium (LD) between markers and genetic variants underlying traits of interest, but the extent of LD in the horse has yet to be comprehensively characterized. In this study, we evaluate the extent and decay of LD in a sample of 817 Thoroughbreds. Horses were genotyped for over 50,000 single nucleotide polymorphism (SNP) markers across the genome, with 34,848 autosomal SNPs used in the final analysis. Linkage disequilibrium, as measured by the squared correlation coefficient (r(2)), was found to be relatively high between closely linked markers (>0.6 at 5 kb) and to extend over long distances, with average r(2) maintained above non-syntenic levels for single nucleotide polymorphisms (SNPs) up to 20 Mb apart. Using formulae which relate expected LD to effective population size (N(e)), and assuming a constant actual population size, N(e) was estimated to be 100 in our population. Values of historical N(e), calculated assuming linear population growth, suggested a decrease in N(e) since the distant past, reaching a minimum twenty generations ago, followed by a subsequent increase until the present time. The qualitative trends observed in N(e) can be rationalized by current knowledge of the history of the Thoroughbred breed, and inbreeding statistics obtained from published pedigree analyses are in agreement with observed values of N(e). Given the high LD observed and the small estimated N(e), genomic methodologies such as genomic selection could feasibly be applied to this population using the existing SNP marker set.

Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58.

Agrobacterium tumefaciens is a plant pathogen capable of transferring a defined segment of DNA to a host plant, generating a gall tumor. Replacing the transferred tumor-inducing genes with exogenous DNA allows the introduction of any desired gene into the plant. Thus, A. tumefaciens has been critical for the development of modern plant genetics and agricultural biotechnology. Here we describe the genome of A. tumefaciens strain C58, which has an unusual structure consisting of one circular and one linear chromosome. We discuss genome architecture and evolution and additional genes potentially involved in virulence and metabolic parasitism of host plants.

Analysis of vertebrate SCL loci identifies conserved enhancers.

The SCL gene encodes a highly conserved bHLH transcription factor with a pivotal role in hemopoiesis and vasculogenesis. We have sequenced and analyzed 320 kb of genomic DNA composing the SCL loci from human, mouse, and chicken. Long-range sequence comparisons demonstrated multiple peaks of human/mouse homology, a subset of which corresponded precisely with known SCL enhancers. Comparisons between mammalian and chicken sequences identified some, but not all, SCL enhancers. Moreover, one peak of human/mouse homology (+23 region), which did not correspond to a known enhancer, showed significant homology to an analogous region of the chicken SCL locus. A transgenic Xenopus reporter assay was established and demonstrated that the +23 region contained a new neural enhancer. This combination of long-range comparative sequence analysis with a high-throughput transgenic bioassay provides a powerful strategy for identifying and characterizing developmentally important enhancers.

Phase Transformations in the High-Tc Superconducting Compounds, Ba2RCu3O7-δ (R = Nd, Sm, Gd, Y, Ho, and Er).

The phase transformation between the orthorhombic and tetragonal structures of six high-T c superconductors, Ba2RCu3O7- δ , where R = Nd, Sm, Gd, Y, Ho, and Er, and δ = 0 to 1, has been investigated using techniques of x-ray diffraction, differential thermal analysis/thermogravimetric analysis (DTA/TGA) and electron diffraction. The transformation from the oxygen-rich orthorhombic phase to the oxygen-deficient tetragonal phase involves two orthorhombic phases. A superlattice cell caused by oxygen ordering, with a' = 2a, was observed for materials with smaller ionic radius (Y, Ho, and Er). For the larger lanthanide samples (Nd, Sm, and Gd), the a' = 2a type superlattice cell was not observed. The structural phase transition temperatures, oxygen stoichiometry and characteristics of the T c plateaus appear to correlate with the ionic radius, which varies based on the number of f electrons. Lanthanide elements with a smaller ionic radius stabilize the orthorhombic phase to higher temperatures and lower oxygen content. Also, the superconducting temperature is less sensitive to the oxygen content for materials with smaller ionic radius. The trend of dependence of the phase transformation temperature on ionic radius across the lanthanide series can be explained using a quasi-chemical approximation (QCA) whereby the strain effect plays an important role on the order-disorder transition due to the effect of oxygen content on the CuO chain sites.

Designing a standard for strain mapping: HR-EBSD analysis of SiGe thin film structures on Si.

Patterned SiGe thin film structures, heteroepitaxially deposited on Si substrates, are investigated as potential reference standards to establish the accuracy of high resolution electron backscattered diffraction (HR-EBSD) strain measurement methods. The proposed standards incorporate thin films of tetragonally distorted epitaxial Si1-xGex adjacent to strain-free Si. Six films of three different nominal compositions (x=0.2, 0.3, and 0.4) and various thicknesses were studied. Film composition and out-of-plane lattice spacing measurements, by x-ray photoelectron spectroscopy and x-ray diffraction, respectively, provided independent determinations of film epitaxy and predictions of tetragonal strain for direct comparison with HR-EBSD strain measurements. Films assessed to be coherent with the substrate exhibited tetragonal strain values measured by HR-EBSD identical to those predicted from the composition and x-ray diffraction measurements, within experimental relative uncertainties of order 2%. Such films thus provide suitable prototypes for designing a strain reference standard.

Tdr2, a new zebrafish transposon of the Tc1 family.

We describe here Tdr2, a new class of Tc1-like transposons in zebrafish. Tdr2 was identified from the genomic sequence of a zebrafish PAC (P1 artificial chromosome) clone, and fragments of Tdr2 were found in several zebrafish EST (expressed sequence tag) sequences. Predicted translation of the Tdr2 transposase gene showed that it was most closely related to Caenorhabditis elegans Tc3A, suggesting an ancient origin of the Tdr2 transposon. Tdr2 spans 1. 1kb and is flanked by inverted repeats of approx. 100bp. The 5' repeat is itself composed of an inverted repeat, raising the possibility of the formation of a cruciform DNA structure. Tdr2 transposons may facilitate the development of novel transposon-based tools for the genetic analysis of zebrafish.

Preparation of hepatitis B polypeptide micelles from human carrier plasma.

Water-soluble protein micelles consisting of the 28,000 (gp28) and 23,00 (p23) molecular weight polypeptide complex of hepatitis B surface antigen can be readily prepared from human plasma containing the surface antigen and other markers of infection with hepatitis B virus. The antigenic activity of the polypeptides was preserved throughout the process of solubilisation and reassociation into micelles. Such preparations are therefore eminently suitable as "second generation" hepatitis B vaccines.

Crystallographic Texture in Ceramics and Metals.

Preferred crystallographic orientation, or texture, occurs almost universally, both in natural and man-made systems. Many components and devices in electronic and magnetic systems are fabricated from materials that have crystallographic texture. With the rapidly increasing use of thin film technology, where sharp axisymmetric crystallographic texture normal to the film plane is frequently observed, the occurrence and impact of texture are rising. Thin film applications in which the texture of the material plays a key role in determining properties and performance are broad: complex oxides in random access memory devices, ZnO thin film resonators for cell phone applications, metallic alloys in magnetic recording media, and Al and Cu interconnects in integrated circuits are but a few examples. Texture is established during the synthesis or post-synthesis heat treatment of a material and thus has a strong dependence upon processing history. Accurate measurement of texture is not simple and a variety of tools and approaches are being actively employed in texture studies. X-ray, neutron and electron diffraction based techniques are practiced around the world at varying levels of complexity with regard to equipment and analysis methods. Despite the well-documented existence of these varied approaches, many reported texture measurements on electronic materials are based solely on the relative intensities of conventional θ-2θ x-ray diffraction peaks, which typically yield inaccurate results. NIST has developed quantitative texture measurement techniques that employ equipment commonly available in most industrial and academic settings. A number of examples of texture measurement in ceramic and metal systems will be presented, taken from the historical development and application of these techniques at NIST over the past 7 years.

Fluorescent and radioactive solid phase dideoxy sequencing of pcr products in microtitre plates.

In this paper we describe a rapid method for the direct generation of DNA sequencing templates from phage or bacteria. Sequencing of these PCR products can be performed by radioactive and fluorescent methods. The non-radioactive method has been used to sequence a total of approximately 100 kb of human DNA fragments generated by digestion with HpaII and subsequent cloning. The method depends on direct small scale amplification using a biotinylated primer, and the binding of the product to streptavidin coated magnetic beads. All the procedures are carried out in a microtitre plate thus facilitating the handling of large numbers of clones and has potential for automation.

High resolution surface morphology measurements using EBSD cross-correlation techniques and AFM.

The surface morphology surrounding wedge indentations in (001) Si has been measured using electron backscattered diffraction (EBSD) and atomic force microscopy (AFM). EBSD measurement of the lattice displacement field relative to a strain-free reference location allowed the surface uplift to be measured by summation of lattice rotations about the indentation axis. AFM was used in intermittent contact mode to determine surface morphology. The height profiles across the indentations for the two techniques agreed within 1 nm. Elastic uplift theory is used to model the data.

Contact-resonance atomic force microscopy for nanoscale elastic property measurements: Spectroscopy and imaging.

Quantitative measurements of the elastic modulus of nanosize systems and nanostructured materials are provided with great accuracy and precision by contact-resonance atomic force microscopy (CR-AFM). As an example of measuring the elastic modulus of nanosize entities, we used the CR-AFM technique to measure the out-of-plane indentation modulus of tellurium nanowires. A size-dependence of the indentation modulus was observed for the investigated tellurium nanowires with diameters in the range 20-150nm. Over this diameter range, the elastic modulus of the outer layers of the tellurium nanowires experienced significant enhancement due to a pronounced surface stiffening effect. Quantitative estimations for the elastic moduli of the outer and inner parts of tellurium nanowires of reduced diameter are made with a core-shell structure model. Besides localized elastic modulus measurements, we have also developed a unique CR-AFM imaging capability to map the elastic modulus over a micrometer-scale area. We used this CR-AFM capability to construct indentation modulus maps at the junction between two adjacent facets of a tellurium microcrystal. The clear contrast observed in the elastic moduli of the two facets indicates the different surface crystallography of these facets.

Canine RPGRIP1 mutation establishes cone-rod dystrophy in miniature longhaired dachshunds as a homologue of human Leber congenital amaurosis.

Cone-rod dystrophy 1 (cord1) is a recessive condition that occurs naturally in miniature longhaired dachshunds (MLHDs). We mapped the cord1 locus to a region of canine chromosome CFA15 that is syntenic with a region of human chromosome 14 (HSA14q11.2) containing the retinitis pigmentosa GTPase regulator-interacting protein 1 (RPGRIP1) gene. Mutations in RPGRIP1 have been shown to cause Leber congenital amaurosis, a group of retinal dystrophies that represent the most common genetic causes of congenital visual impairment in infants and children. Using the newly available canine genome sequence we sequenced RPGRIP1 in affected and carrier MLHDs and identified a 44-nucleotide insertion in exon 2 that alters the reading frame and introduces a premature stop codon. All affected and carrier dogs within an extended inbred pedigree were homozygous and heterozygous, respectively, for the mutation. We conclude the mutation is responsible for cord1 and demonstrate that this canine disease is a valuable model for exploring disease mechanisms and potential therapies for human Leber congenital amaurosis.

Evolution of sensory complexity recorded in a myxobacterial genome.

Myxobacteria are single-celled, but social, eubacterial predators. Upon starvation they build multicellular fruiting bodies using a developmental program that progressively changes the pattern of cell movement and the repertoire of genes expressed. Development terminates with spore differentiation and is coordinated by both diffusible and cell-bound signals. The growth and development of Myxococcus xanthus is regulated by the integration of multiple signals from outside the cells with physiological signals from within. A collection of M. xanthus cells behaves, in many respects, like a multicellular organism. For these reasons M. xanthus offers unparalleled access to a regulatory network that controls development and that organizes cell movement on surfaces. The genome of M. xanthus is large (9.14 Mb), considerably larger than the other sequenced delta-proteobacteria. We suggest that gene duplication and divergence were major contributors to genomic expansion from its progenitor. More than 1,500 duplications specific to the myxobacterial lineage were identified, representing >15% of the total genes. Genes were not duplicated at random; rather, genes for cell-cell signaling, small molecule sensing, and integrative transcription control were amplified selectively. Families of genes encoding the production of secondary metabolites are overrepresented in the genome but may have been received by horizontal gene transfer and are likely to be important for predation.

Mapping nonisotopically labeled DNA probes to human chromosome bands by confocal microscopy.

A method for mapping nonisotopically labeled probes to human metaphase chromosomes that can be used with laser scanning confocal microscopy has been developed. Only a limited number of wavelengths are available from the argon ion lasers used in most commercial instruments and therefore a method that allowed the visualization of bands on human chromosomes stained with propidium iodide and, simultaneously, the detection of hybridization signals using FITC-labeled antibodies was developed. The confocal microscope was used to map single-copy probes to chromosome bands and the positions of the probes on the R-banded chromosomes corresponded to map positions previously determined on Hoechst 33258-stained chromosomes (G-banded). A comparison of confocal imaging of single-copy hybridization signals with conventional fluorescence microscopy and high-sensitivity video cameras revealed little difference in sensitivity but greater resolution of chromosome bands with the confocal microscope. The polymerase chain reaction was used to prepare nonisotopically labeled probes for in situ hybridization and to amplify Alu and KpnI family repeats from cloned DNA to be used to suppress hybridization of these repeat sequences so that a cosmid probe could be mapped to a chromosome band.

The complete nucleotide sequence of the genome of a hepatitis B virus isolated from a naturally infected chimpanzee.

The complete nucleotide sequence of a strain of hepatitis B virus, originally isolated from a naturally infected chimpanzee, has been determined. Interesting features of the sequence include the presence of an in-phase stop codon in the 'pre-core' region of the core antigen open reading frame. The sequence shows approximately 10% nucleotide divergence from all of the other hepatitis B virus sequences previously published and the possibility that this divergence is the result of passage through chimpanzees is discussed.