Construction of a bacterial artificial chromosome (BAC) library of Lycopersicon esculentum cv. Stevens and its application to physically map the Sw-5 locus

The Sw-5 gene is a dominantly inherited resistance gene in tomato and functional against a number of tospovirus species. The gene has been mapped on chromosome 9, tightly linked to RFLP markers CT220 and SCAR421. To analyse the Sw-5 locus, a BAC genomic library was constructed of tomato cv. Stevens, homozygous for the Sw-5 gene. The library comprised 18 816 clones with an average insert size of 100 kb, corresponding to two genome equivalents. The library was screened by PCR using primers designed for the CT220 and SCAR421 sequences, resulting in a 250 kb contig of known orientation on the long arm of chromosome 9. Using degenerate primers based on homologous sequences in the nucleotide binding site of resistance gene sequences, three discrete PCR fragments obtained from this contig were cloned and sequenced. Analysis of these fragments revealed a high similarity with numerous resistance genes or resistance gene like sequences. The present data indicate that at least three different resistance gene candidate (RGC) sequences are present in the vicinity of marker CT220, supporting the view that a resistance gene family may be responsible for the unusually broad resistance to tospoviruses conferred by the Sw-5 locus. This revised version was published online in June 2006 with corrections to the Cover Date.     

Association of Hereditary Nonpolyposis Colorectal Cancer–Related Tumors Displaying Low Microsatellite Instability with MSH6 Germline Mutations

Hereditary nonpolyposis colorectal cancer (HNPCC) (Amsterdam criteria) is often caused by mutations in mismatch repair (MMR) genes, and tumors of patients with HNPCC show microsatellite instability (MSI-high phenotype). Germline mutations of MMR genes have rarely been found in families that have HNPCC or suspected HNPCC and that do not show microsatellite instability (MSI-low phenotype). Therefore, an MSI-high phenotype is often used as an inclusion criterion for mutation testing of MMR genes. Correction of base-base mismatches is the major function of MSH6. Since mismatches present with an MSI-low phenotype, we assumed that the phenotype in patients with HNPCC-related tumors might be associated with MSH6 germline mutations. We divided 36 patients with suspected HNPCC into an MSI-low group (n=18) and an MSI-high group (n=18), on the basis of the results of MSI testing. Additionally, three unrelated patients from Amsterdam families with MSI-low tumors were investigated. All patients were screened for MSH2, MLH1, and MSH6 mutations. Four presumably causative MSH6 mutations were detected in the patients (22%) who had suspected HNPCC and MSI-low tumors. Furthermore, we detected one frameshift mutation in one of the three patients with HNPCC and MSI-low tumors. In the MSI-high group, one MSH6 missense mutation was found, but the same patient also had an MLH1 mutation, which may explain the MSI-high phenotype. These results suggest that MSH6 may be involved in a substantial proportion of patients with HNPCC or suspected HNPCC and MSI-low tumors. Our data emphasize that an MSI-low phenotype cannot be considered an exclusion criterion for mutation testing of MMR genes in general.     

Microsatellite repeat instability and neurological disease

Over 20 unstable microsatellite repeats have been identified as the cause of neurological disease in humans. The repeat nucleotide sequences, their location within the genes, the ranges of normal and disease‐causing repeat length and the clinical outcomes differ. Unstable repeats can be located in the coding or the non‐coding region of a gene. Different pathogenic mechanisms that are hypothesised to underlie the diseases are discussed. Evidence is given both from studies in simple model systems and from studies on human material and in animal models. Since somatic instability might affect the clinical outcome, this is briefly touched on. Available data and theories on the timing and mechanisms of the repeat instability itself are discussed, along with factors that have been observed to affect instability. Finally, the question of why the often harmful unstable repeats have been maintained throughout evolution is addressed.     

Modeling Basic Industries in the Australian Stocks and Flows Framework

The Australian stocks and flows framework (ASFF) is a tool for establishing a coherent historical picture of the Australian physical economy and for testing long-term future scenarios (up to 2050 or even 2100). These scenarios can be used to investigate the long-term physical consequences of current and future choices affecting the physical dimensions of sustainability. In this article we describe the methodology for and construction of a key component of ASFF: a dynamic physical input-output model of material flows in the basic industries.
The materials model in ASFF describes physical flows and their transformation by industrial processes. The model's structure permits scenario analysis of long-term technological change by permitting time-varying input-output coefficients and vintage models of capital stocks. As a consequence, the model contains a large number of parameters, which can be left at default settings or adjusted as the modeler sees fit, in order to simulate the widest possible range of physically realizable scenarios. The materials model is built using a methodology that integrates bottom-up process analysis with top-down statistics on material and energy flows. We present some examples showing how the materials model has been implemented to model Australian heavy industries. Several possibilities for further developing the materials model are also described.     

Water and deuterium oxide permeability through aquaporin 1: MD predictions and experimental verification

Determining the mechanisms of flux through protein channels requires a combination of structural data, permeability measurement, and molecular dynamics (MD) simulations. To further clarify the mechanism of flux through aquaporin 1 (AQP1), osmotic p(f) (cm(3)/s/pore) and diffusion p(d) (cm(3)/s/pore) permeability coefficients per pore of H(2)O and D(2)O in AQP1 were calculated using MD simulations. We then compared the simulation results with experimental measurements of the osmotic AQP1 permeabilities of H(2)O and D(2)O. In this manner we evaluated the ability of MD simulations to predict actual flux results. For the MD simulations, the force field parameters of the D(2)O model were reparameterized from the TIP3P water model to reproduce the experimentally observed difference in the bulk self diffusion constants of H(2)O vs. D(2)O. Two MD systems (one for each solvent) were constructed, each containing explicit palmitoyl-oleoyl-phosphatidyl-ethanolamine (POPE) phospholipid molecules, solvent, and AQP1. It was found that the calculated value of p(f) for D(2)O is approximately 15% smaller than for H(2)O. Bovine AQP1 was reconstituted into palmitoyl-oleoyl-phosphatidylcholine (POPC) liposomes, and it was found that the measured macroscopic osmotic permeability coefficient P(f) (cm/s) of D(2)O is approximately 21% lower than for H(2)O. The combined computational and experimental results suggest that deuterium oxide permeability through AQP1 is similar to that of water. The slightly lower observed osmotic permeability of D(2)O compared to H(2)O in AQP1 is most likely due to the lower self diffusion constant of D(2)O.