Centromere mapping and orientation of the molecular linkage map of rice (Oryza sativa L.).

Rice has become a model cereal plant for molecular genetic research. Rice has the most comprehensive molecular linkage maps with more than 2000 DNA markers and shows synteny and colinearity with the maps of other cereal crops. Until now, however, no information was available about the positions of centromeres and arm locations of markers on the molecular linkage map. Secondary and telotrisomics were used to assign restriction fragment length polymorphism markers to specific chromosome arms and thereby to map the positions of centromeres. More than 170 restriction fragment length polymorphism markers were assigned to specific chromosome arms through gene dosage analysis using the secondary and telotrisomics and the centromere positions were mapped on all 12 linkage groups. The orientations of seven linkage groups were reversed to fit the "short arm on top" convention and the corrected map is presented.

A rat genetic map constructed by representational difference analysis markers with suitability for large-scale typing.

Representational difference analysis (RDA) was applied to isolate chromosomal markers in the rat. Four series of RDA [restriction enzymes, BamHI and HindIII; subtraction of ACI/N (ACI) amplicon from BUF/Nac (BUF) amplicon and vice versa] yielded 131 polymorphic markers; 125 of these markers were mapped to all chromosomes except for chromosome X. This was done by using a mapping panel of 105 ACI x BUF F2 rats. To complement the relative paucity of chromosomal markers in the rat, genetically directed RDA, which allows isolation of polymorphic markers in the specific chromosomal region, was performed. By changing the F2 driver-DNA allele frequency around the region, four markers were isolated from the D1Ncc1 locus. Twenty-five of 27 RDA markers were informative regarding the dot blot analysis of amplicons, hybridizing only with tester amplicons. Dot blot analysis at a high density per unit of area made it possible to process a large number of samples. Quantitative trait loci can now be mapped in the rat genome by processing a large number of samples with RDA markers and then by isolating markers close to the loci of interest by genetically directed RDA.

A yeast artificial chromosome-based map of the region of chromosome 20 containing the diabetes-susceptibility gene, MODY1, and a myeloid leukemia related gene.

We have generated a physical map of human chromosome bands 20q11.2-20q13.1, a region containing a gene involved in the development of one form of early-onset, non-insulin-dependent diabetes mellitus, MODY1, as well as a putative myeloid tumor suppressor gene. The yeast artificial chromosome contig consists of 71 clones onto which 71 markers, including 20 genes, 5 expressed sequence tags, 32 simple tandem repeat DNA polymorphisms, and 14 sequence-tagged sites have been ordered. This region spans about 18 Mb, which represents about 40% of the physical length of 20q. Using this physical map, we have refined the location of MODY1 to a 13-centimorgan interval (approximately equal to 7 Mb) between D20S169 and D20S176. The myeloid tumor suppressor gene was localized to an 18-centimorgan interval (approximately equal to 13 Mb) between RPN2 and D20S17. This physical map will facilitate the isolation of MODY1 and the myeloid tumor suppressor gene.

High-resolution physical mapping by combined Alu-hybridization/PCR screening: construction of a yeast artificial chromosome map covering 31 centimorgans in 3p21-p14.

We describe an integrated approach to large-scale physical mapping using an Alu-PCR hybridization screening strategy in conjunction with direct PCR-based screening to construct a continuous yeast artificial chromosome map covering >20 mb in human chromosome 3, bands p14-p21, composed of 205 loci, connected by 480 yeast artificial chromosomes, with average interlocus distance of approximately equal to 100 kb. We observe an inverse distribution of Alu-PCR and (CA)n markers. These results suggest that the two screening methods may be complementary and demonstrate the utility of Alu-PCR hybridization screening in the closure of high-resolution human physical maps.

Neurotrophins stimulate phosphorylation of synapsin I by MAP kinase and regulate synapsin I-actin interactions.

The ability of neurotrophins to modulate the survival and differentiation of neuronal populations involves the Trk/MAP (mitogen-activated protein kinase) kinase signaling pathway. More recently, neurotrophins have also been shown to regulate synaptic transmission. The synapsins are a family of neuron-specific phosphoproteins that play a role in regulation of neurotransmitter release, in axonal elongation, and in formation and maintenance of synaptic contacts. We report here that synapsin I is a downstream effector for the neurotrophin/Trk/MAP kinase cascade. Using purified components, we show that MAP kinase stoichiometrically phosphorylated synapsin I at three sites (Ser-62, Ser-67, and Ser-549). Phosphorylation of these sites was detected in rat brain homogenates, in cultured cerebrocortical neurons, and in isolated presynaptic terminals. Brain-derived neurotrophic factor and nerve growth factor upregulated phosphorylation of synapsin I at MAP kinase-dependent sites in intact cerebrocortical neurons and PC12 cells, respectively, while KCl- induced depolarization of cultured neurons decreased the phosphorylation state at these sites. MAP kinase-dependent phosphorylation of synapsin I significantly reduced its ability to promote G-actin polymerization and to bundle actin filaments. The results suggest that MAP kinase-dependent phosphorylation of synapsin I may contribute to the modulation of synaptic plasticity by neurotrophins and by other signaling pathways that converge at the level of MAP kinase activation.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae.

An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis, was constructed by using a twin-pronged approach. Pulsed-field gel electrophoretic analysis enabled cleavage sites for Asn I and Dra I to be positioned on the 4.4-Mb circular chromosome, while, in parallel, clones from two cosmid libraries were ordered into contigs by means of fingerprinting and hybridization mapping. The resultant contig map was readily correlated with the physical map of the genome via the landmarked restriction sites. Over 165 genes and markers were localized on the integrated map, thus enabling comparisons with the leprosy bacillus, Mycobacterium leprae, to be undertaken. Mycobacterial genomes appear to have evolved as mosaic structures since extended segments with conserved gene order and organization are interspersed with different flanking regions. Repetitive sequences and insertion elements are highly abundant in M. tuberculosis, but the distribution of IS6110 is apparently nonrandom.

A high-resolution physical map of human chromosome 11.

The development of a highly reliable physical map with landmark sites spaced an average of 100 kbp apart has been a central goal of the Human Genome Project. We have approached the physical mapping of human chromosome 11 with this goal as a primary target. We have focused on strategies that would utilize yeast artificial chromosome (YAC) technology, thus permitting long-range coverage of hundreds of kilobases of genomic DNA, yet we sought to minimize the ambiguities inherent in the use of this technology, particularly the occurrence of chimeric genomic DNA clones. This was achieved through the development of a chromosome 11-specific YAC library from a human somatic cell hybrid line that has retained chromosome 11 as its sole human component.To maximize the efficiency of YAC contig assembly and extension, we have employed an Alu-PCR-based hybridization screening system. This system eliminates many of the more costly and time-consuming steps associated with sequence tagged site content mapping such as sequencing, primer production, and hierarchical screening, resulting in greater efficiency with increased throughput and reduced cost. Using these approaches, we have achieved YAC coverage for >90% of human chromosome 11, with an average intermarker distance of <100 kbp. Cytogenetic localization has been determined for each contig by fluorescent in situ hybridization and/or sequence tagged site content. The YAC contigs that we have generated should provide a robust framework to move forward to sequence-ready templates for the sequencing efforts of the Human Genome Project as well as more focused positional cloning on chromosome 11.

A compositional map of human chromosome band Xq28.

The molar fractions of guanine plus cytosine (GC) in DNA were determined for 36 yeast artificial chromosomes (YACs) which almost completely cover human chromosome band Xq28, a terminal reverse band, corresponding to about 8 Mb of DNA. This allowed the construction of the most complete compositional map to date of a chromosomal band; three regions were observed: (i) a proximal 3.5-Mb region formed by GC-poor L and GC-rich H1 isochores; (ii) a middle 2,2-Mb region essentially formed by a GC-rich H2 isochore and a very GC-rich H3 isochore separated by a GC-poor L isochore, YACs from this region being characterized by a striking compositional heterogeneity and instability; and (iii) a distal 1.3-Mb region exclusively formed by GC-poor L isochores. Gene and CpG island concentrations increased with the GC levels of the isochores, as expected. Xq28 exemplifies a subset of reverse bands which are different from the two other subsets, namely from telomeric bands, which are characterized by specific cytogenetic properties and by the predominance of H2 and H3 isochores, and from the majority of reverse bands, which do not contain H2 and H3 isochores.

A high-resolution annotated physical map of the human chromosome 13q12-13 region containing the breast cancer susceptibility locus BRCA2.

Various types of physical mapping data were assembled by developing a set of computer programs (Integrated Mapping Package) to derive a detailed, annotated map of a 4-Mb region of human chromosome 13 that includes the BRCA2 locus. The final assembly consists of a yeast artificial chromosome (YAC) contig with 42 members spanning the 13q12-13 region and aligned contigs of 399 cosmids established by cross-hybridization between the cosmids, which were selected from a chromosome 13-specific cosmid library using inter-Alu PCR probes from the YACs. The end sequences of 60 cosmids spaced nearly evenly across the map were used to generate sequence-tagged sites (STSs), which were mapped to the YACs by PCR. A contig framework was generated by STS content mapping, and the map was assembled on this scaffold. Additional annotation was provided by 72 expressed sequences and 10 genetic markers that were positioned on the map by hybridization to cosmids.

Parvovirus B19 promoter at map unit 6 confers autonomous replication competence and erythroid specificity to adeno-associated virus 2 in primary human hematopoietic progenitor cells.

The pathogenic human parvovirus B19 is an autonomously replicating virus with a remarkable tropism for human erythroid progenitor cells. Although the target cell specificity for B19 infection has been suggested to be mediated by the erythrocyte P-antigen receptor (globoside), a number of nonerythroid cells that express this receptor are nonpermissive for B19 replication. To directly test the role of expression from the B19 promoter at map unit 6 (B19p6) in the erythroid cell specificity of B19, we constructed a recombinant adeno-associated virus 2 (AAV), in which the authentic AAV promoter at map unit 5 (AAVp5) was replaced by the B19p6 promoter. Although the wild-type (wt) AAV requires a helper virus for its optimal replication, we hypothesized that inserting the B19p6 promoter in a recombinant AAV would permit autonomous viral replication, but only in erythroid progenitor cells. In this report, we provide evidence that the B19p6 promoter is necessary and sufficient to impart autonomous replication competence and erythroid specificity to AAV in primary human hematopoietic progenitor cells. Thus, expression from the B19p6 promoter plays an important role in post-P-antigen receptor erythroid-cell specificity of parvovirus B19. The AAV-B19 hybrid vector system may also prove to be useful in potential gene therapy of human hemoglobinopathies.

Genomic structure of human microtubule-associated protein 2 (MAP-2) and characterization of additional MAP-2 isoforms.

We have determined that the gene for human microtubule-associated protein 2 (MAP-2) spans 19 exons, including 6 exons identified in this study, 1-4, 8, and 13; all six of these exons are transcribed. The alternative splicing of coding exons generates a greater diversity of MAP-2 transcripts and isoforms. The first three exons encode alternate 5' untranslated regions that can be spliced to additional untranslated sequences contained in exons 4 and 5. Exons 8 and 13 are transcribed in human fetal spinal cord, adult brain, MSN cells, and rat brain, and each exon maintains an open reading frame with both high and low molecular weight MAP-2 isoforms. Antibodies generated to synthetic peptides of exons 8 and 13 demonstrate that these exons are translated and MAP-2 isoforms containing these exons are generated.

Transmission and Transport of Energy in the Western U.S. and Canada: A Law and Policy Road Map

This collection of short essays arose from the inaugural meeting of the Idaho Symposium on Energy in the West, which was held in November, 2014. The topic for this first Symposium was Transmission and Transport of Energy in the Western U.S. and Canada: A Law and Policy Road Map. The essays in this collection provide a notable introduction to the major energy issues facing the West today. Topics include: building a resilient legal architecture for western energy production; natural gas flaring; transmission planning for wind energy; utilities and rooftop solar; special considerations for western states and the Clean Power Plan; the Clean Power Plan's implications for the western grid; siting renewable energy on public lands; and implications of utility reform in New York and Hawaii for the Northwest.

3D model based map building

Comunicación presentada en el IX Workshop de Agentes Físicos (WAF'2008), Vigo, 11-12 septiembre 2008.

Ocular residual astigmatism and topographic disparity vector indexes in normal healthy eyes

Purpose: To define a range of normality for the vectorial parameters Ocular Residual Astigmatism (ORA) and topography disparity (TD) and to evaluate their relationship with visual, refractive, anterior and posterior corneal curvature, pachymetric and corneal volume data in normal healthy eyes. Methods: This study comprised a total of 101 consecutive normal healthy eyes of 101 patients ranging in age from 15 to 64 years old. In all cases, a complete corneal analysis was performed using a Scheimpflug photography-based topography system (Pentacam system Oculus Optikgeräte GmbH). Anterior corneal topographic data were imported from the Pentacam system to the iASSORT software (ASSORT Pty. Ltd.), which allowed the calculation of the ocular residual astigmatism (ORA) and topography disparity (TD). Linear regression analysis was used for obtaining a linear expression relating ORA and posterior corneal astigmatism (PCA). Results: Mean magnitude of ORA was 0.79 D (SD: 0.43), with a normality range from 0 to 1.63 D. 90 eyes (89.1%) showed against-the-rule ORA. A weak although statistically significant correlation was found between the magnitudes of posterior corneal astigmatism and ORA (r = 0.34, p < 0.01). Regression analysis showed the presence of a linear relationship between these two variables, although with a very limited predictability (R2: 0.08). Mean magnitude of TD was 0.89 D (SD: 0.50), with a normality range from 0 to 1.87 D. Conclusion: The magnitude of the vector parameters ORA and TD is lower than 1.9 D in the healthy human eye.

Clinical utility of ocular residual astigmatism and topographic disparity vector indexes in subclinical and clinical keratoconus

Purpose. We aimed to characterize the distribution of the vector parameters ocular residual astigmatism (ORA) and topography disparity (TD) in a sample of clinical and subclinical keratoconus eyes, and to evaluate their diagnostic value to discriminate between these conditions and healthy corneas. Methods. This study comprised a total of 43 keratoconic eyes (27 patients, 17–73 years) (keratoconus group), 11 subclinical keratoconus eyes (eight patients, 11–54 years) (subclinical keratoconus group) and 101 healthy eyes (101 patients, 15–64 years) (control group). In all cases, a complete corneal analysis was performed using a Scheimpflug photography-based topography system. Anterior corneal topographic data was imported from it to the iASSORT software (ASSORT Pty. Ltd), which allowed the calculation of ORA and TD. Results. Mean magnitude of the ORA was 3.23 ± 2.38, 1.16 ± 0.50 and 0.79 ± 0.43 D in the keratoconus, subclinical keratoconus and control groups, respectively (p < 0.001). Mean magnitude of the TD was 9.04 ± 8.08, 2.69 ± 2.42 and 0.89 ± 0.50 D in the keratoconus, subclinical keratoconus and control groups, respectively (p < 0.001). Good diagnostic performance of ORA (cutoff point: 1.21 D, sensitivity 83.7 %, specificity 87.1 %) and TD (cutoff point: 1.64 D, sensitivity 93.3 %, specificity 92.1 %) was found for the detection of keratoconus. The diagnostic ability of these parameters for the detection of subclinical keratoconus was more limited (ORA: cutoff 1.17 D, sensitivity 60.0 %, specificity 84.2 %; TD: cutoff 1.29 D, sensitivity 80.0 %, specificity 80.2 %). Conclusion. The vector parameters ORA and TD are able to discriminate with good levels of precision between keratoconus and healthy corneas. For the detection of subclinical keratoconus, only TD seems to be valid.