Development of a pooled probe method for locating small gene families in a physical map of soybean using stress related paralogues and a BAC minimum tile path

BACKGROUND: Genome analysis of soybean (Glycine max L.) has been complicated by its paleo-autopolyploid nature and conserved homeologous regions. Landmarks of expressed sequence tags (ESTs) located within a minimum tile path (MTP) of contiguous (contig) bacterial artificial chromosome (BAC) clones or radiation hybrid set can identify stress and defense related gene rich regions in the genome. A physical map of about 2,800 contigs and MTPs of 8,064 BAC clones encompass the soybean genome. That genome is being sequenced by whole genome shotgun methods so that reliable estimates of gene family size and gene locations will provide a useful tool for finishing. The aims here were to develop methods to anchor plant defense- and stress-related gene paralogues on the MTP derived from the soybean physical map, to identify gene rich regions and to correlate those with QTL for disease resistance. RESULTS: The probes included 143 ESTs from a root library selected by subtractive hybridization from a multiply disease resistant soybean cultivar 'Forrest' 14 days after inoculation with Fusarium solani f. sp. glycines (F. virguliforme). Another 166 probes were chosen from a root EST library (Gm-r1021) prepared from a non-inoculated soybean cultivar 'Williams 82' based on their homology to the known defense and stress related genes. Twelve and thirteen pooled EST probes were hybridized to high-density colony arrays of MTP BAC clones from the cv. 'Forrest' genome. The EST pools located 613 paralogues for 201 of the 309 probes used (range 1–13 per functional probe). One hundred BAC clones contained more than one kind of paralogue. Many more BACs (246) contained a single paralogue of one of the 201 probes detectable gene families. ESTs were anchored on soybean linkage groups A1, B1, C2, E, D1a+Q, G, I, M, H, and O. CONCLUSION: Estimates of gene family sizes were more similar to those made by Southern hybridization than by bioinformatics inferences from EST collections. When compared to Arabidopsis thaliana there were more 2 and 4 member paralogue families reflecting the diploidized-tetraploid nature of the soybean genome. However there were fewer families with 5 or more genes and the same number of single genes. Therefore the method can identify evolutionary patterns such as massively extensive selective gene loss or rapid divergence to regenerate the unique genes in some families

Electrical Conductivity of Fermi Liquids. I. Many-body Effect on the Drude Weight

On the basis of the Fermi liquid theory, we investigate the many-body effect on the Drude weight. In a lattice system, the Drude weight $D$ is modified by electron-electron interaction due to Umklapp processes, while it is not renormalized in a Galilean invariant system. This is explained by showing that the effective mass $m'$ for $D\propto n/m'$ is defined through the current, not velocity, of quasiparticle. It is shown that the inequality $D>0$ is required for the stability against the uniform shift of the Fermi surface. The result of perturbation theory applied for the Hubbard model indicates that $D$ as a function of the density $n$ is qualitatively modified around half filling $n\sim 1$ by Umklapp processes.Comment: 20 pages, 2 figures; J. Phys. Soc. Jpn. Vol.67, No.

Poly(styrene-co-vinylbenzylchloride-co-divinylbenzene) coated iron oxide: Synthesis and effects on size and morphology

Iron oxide nanoparticles were coated with a polymer synthesized from the monomers of styrene, divinylbenzene, and vinylbenzylchloride. The resultant polymer microspheres from synthesis without nanoparticle loading are primarily monodispersed with a diameter of 3.45 μm as measured by scanning electron microscopy. The addition of 1% nanoparticles by mass appears to decrease the size of the microspheres down to 2.04 μm as well as increase the polydispersity. This trend is also seen to continue as you add more nanoparticles to the system going from 3.45 μm with 0% nanoparticles down to below 1 μm for 5% nanoparticles. This indicates that the particles are not just incorporated into the polymer matrix but act as nucleation sites to begin the polymerization process. The polymerization process was found to have no effect on the nanoparticles themselves as the magnetic characterization showed only a mass dilution in saturation when corrected by thermal gravimetric analysis

Patient Compliance With Follow-Up After Open Reduction and Internal Fixation for Treating Malleolar Ankle Fractures: A Retrospective Review

Background: Compliance with follow-up after orthopaedic procedures is variable and does not always occur as recommended. Various factors such as medical, financial, cultural, and logistical reasons may contribute to this lack of compliance. The purpose of this study was to determine follow-up compliance of patients who had undergone open reduction and internal fixation (ORIF) for treating closed malleolar ankle fractures. Methods: Medical records of patients who underwent ORIF for treating closed malleolar ankle fractures by the senior author (RAM) were reviewed to evaluate compliance with postoperative follow-up (n = 267). Inclusion criteria were patients with isolated, acute, closed fractures (n = 229). Patients were considered to have followed up appropriately if they returned to clinic after a removable cast boot was issued at 4 to 8 weeks postoperatively. A 2-tailed t test was performed to analyze age and visual analogue scale score at the time of obtaining the removable cast boot. Chi-square testing was performed to analyze the other variables studied. Results: Of the 229 patients included, a total of 183 complied with follow-up whereas 46 did not. Younger age, male sex, and living greater than 160.9 km (100 mi) from the hospital were statistically significant variables associated with decreased compliance with follow-up. Conclusions: In our patient population, 80% of patients followed up in clinic as scheduled. The remaining 20% did not adhere with scheduled followup either before or after obtaining a removable cast boot. Younger age, male sex, and living greater than 100 miles from the hospital were associated with decreased compliance. Consideration should be paid to these factors when treating patients with ankle fractures

Research in and application of modern automatic control theory to nuclear rocket dynamics and control, volume I Semiannual status report

Linear optimal feedback control theory for nuclear rocket dynamics and control problem

Fine map of the Gct1 spontaneous ovarian granulosa cell tumor locus

The spontaneous development of juvenile-onset, ovarian granulosa cell (GC) tumors in the SWR/Bm (SWR) inbred mouse strain is a model for juvenile-type GC tumors that appear in infants and young girls. GC tumor susceptibility is supported by multiple Granulosa cell tumor (Gct) loci, but the Gct1 locus on Chr 4 derived from SWR strain background is fundamental for GC tumor development and uniquely responsive to the androgenic precursor dehydroepiandrosterone (DHEA). To resolve the location of Gct1 independently from other susceptibility loci, Gct1 was isolated in a congenic strain that replaces the distal segment of Chr 4 in SWR mice with a 47 × 10(6)-bp genomic segment from the Castaneus/Ei (CAST) strain. SWR females homozygous for the CAST donor segment were confirmed to be resistant to DHEA- and testosterone-induced GC tumorigenesis, indicating successful exchange of CAST alleles (Gct1(CA)) for SWR alleles (Gct1(SW)) at this tumor susceptibility locus. A series of nested, overlapping, congenic sublines was created to fine-map Gct1 based on GC tumor susceptibility under the influence of pubertal DHEA treatment. Twelve informative lines have resolved the Gct1 locus to a 1.31 × 10(6)-bp interval on mouse Chr 4, a region orthologous to human Chr 1p36.22. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00335-012-9439-6) contains supplementary material, which is available to authorized users

A Novel Bioluminescent Protease Assay Using Engineered Firefly Luciferase

Proteases play important roles in a variety of disease processes. Understanding their biological functions underpins the efforts of drug discovery. We have developed a bioluminescent protease assay using a circularly permuted form of firefly luciferase, wherein the native enzyme termini were joined by a peptide containing a protease site of interest. Protease cleavage of these mutant luciferases greatly activates the enzyme, typically over 100 fold. The mutant luciferase substrates are easily generated by molecular cloning and cell-free translation reactions and thus the protease substrates do not need to be chemically synthesized or purchased. The assay has broad applicability using a variety of proteases and their cognate sites and can sensitively detect protease activity. In this report we further demonstrate its utility for the evaluation of protease recognition sequence specificity and subsequent establishment of an optimized assay for the identification and characterization of protease inhibitors using high throughput screening

HLA-A2–Matched Peripheral Blood Mononuclear Cells From Type 1 Diabetic Patients, but Not Nondiabetic Donors, Transfer Insulitis to NOD-scid/γcnull/HLA-A2 Transgenic Mice Concurrent With the Expansion of Islet-Specific CD8+ T cells

OBJECTIVE: Type 1 diabetes is an autoimmune disease characterized by the destruction of insulin-producing beta-cells. NOD mice provide a useful tool for understanding disease pathogenesis and progression. Although much has been learned from studies with NOD mice, increased understanding of human type 1 diabetes can be gained by evaluating the pathogenic potential of human diabetogenic effector cells in vivo. Therefore, our objective in this study was to develop a small-animal model using human effector cells to study type 1 diabetes. RESEARCH DESIGN AND METHODS: We adoptively transferred HLA-A2-matched peripheral blood mononuclear cells (PBMCs) from type 1 diabetic patients and nondiabetic control subjects into transgenic NOD-scid/gammac(null)/HLA-A*0201 (NOD-scid/gammac(null)/A2) mice. At various times after adoptive transfer, we determined the ability of these mice to support the survival and proliferation of the human lymphoid cells. Human lymphocytes were isolated and assessed from the blood, spleen, pancreatic lymph node and islets of NOD-scid/gammac(null)/A2 mice after transfer. RESULTS: Human T and B cells proliferate and survive for at least 6 weeks and were recovered from the blood, spleen, draining pancreatic lymph node, and most importantly, islets of NOD-scid/gammac(null)/A2 mice. Lymphocytes from type 1 diabetic patients preferentially infiltrate the islets of NOD-scid/gammac(null)/A2 mice. In contrast, PBMCs from nondiabetic HLA-A2-matched donors showed significantly less islet infiltration. Moreover, in mice that received PBMCs from type 1 diabetic patients, we identified epitope-specific CD8(+) T cells among the islet infiltrates. CONCLUSIONS: We show that insulitis is transferred to NOD-scid/gammac(null)/A2 mice that received HLA-A2-matched PBMCs from type 1 diabetic patients. In addition, many of the infiltrating CD8(+) T cells are epitope-specific and produce interferon-gamma after in vitro peptide stimulation. This indicates that NOD-scid/gammac(null)/A2 mice transferred with HLA-A2-matched PBMCs from type 1 diabetic patients may serve as a useful tool for studying epitope-specific T-cell-mediated responses in patients with type 1 diabetes