Use of the additive main effects and multiplicative interaction model in QTL mapping for adaptation in barley

The additive main effects and multiplicative interaction (AMMI) model has emerged as a powerful analytical tool for genotype x environment studies. The objective of the present study was to assess its value in quantitative trait locus (QTL) mapping. This was done through the analysis of a large two-way table of genotype-by-environment data of barley (Hordeum vulgare L.) grain yields, where the genotypes constituted a genetic population suitable for mapping studies. Grain yield data of 150 doubled haploid lines derived from the Steptoe x Morex cross, and the two parental lines, were taken by the North American Barley Genome Mapping Project (NABGMP) at 16 environments throughout the barley production areas of the USA and Canada. Four regions of the genome were responsible for most of the differential genotypic expression across environments. They accounted for approximately 50% of the genotypic main effect and 30% of the genotype x environment interaction (GE) sums of squares. The magnitude and sign of AMMI scores for genotypes and sites facilitate inferences about specific interactions. The parallel use of classification (cluster analysis of environments) and ordination (principal component analysis of GE matrix) techniques allowed most of the variation present in the genotype x environment matrix to be summarized in just a few dimensions, specifically four QTLs showing differential adaptation to four clusters of environments. Thus, AMMI genotypic scores, when the genotypes constituted a population suitable for QTL mapping, could provide an adequate way of resolving the magnitude and nature of QTL x environment interactions.Ignacio Romagosa was on sabbatical leave from the University of Lleida and the Institut de Recerca i Tecnologia Agroalimentàries, Lleida, Spain, when this study was conducted     

The Mouse Glutathione PeroxidaseGpx2Gene Maps to Chromosome 12; Its PseudogeneGpx2-psMaps to Chromosome 7

TheGPX2gene codes for GSHPx-GI, a glutathione peroxidase whose mRNA is readily detectable in the gastrointestinal tract. AlthoughGPX2is a single gene in humans, there are two genes in the mouse genome with homology toGPX2.By analyzing a panel of mouse interspecies DNA from the Jackson Laboratory's backcross resource, we have chromosomally mapped these two genes. One was mapped to the central region of mouse chromosome 12 betweenD12Mit4andD12Mit5,nearfosandTgfb3.This region is homologous to human 14q24.1, where humanGPX2has been mapped, and most likely represents the functional mouseGpx2gene. The otherGpx2-like gene was mapped to mouse chromosome 7 betweenPcsk3andHbb.We have isolated the latter gene from a P1 phage library. Its pseudogene nature is revealed by the sequence analysis: (a) it is intronless; (b) it has a single nucleotide deletion in the coding region; and (c) it has a poly(A) tail at its 3′-untranslated region.     

The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.     

An improved method for the isolation of type II and clara cells from mice

Identifying the causal events and temporal aspects of lung cancer development requires the ability to isolate target and nontarget cells for comparative analyses. Current methodology can either isolate only one pure specific cell population from a lung or multiple cell types at lower purity. Previous studies in our laboratory have identified the alveolar type II cell as the progenitor cell for tumor development in the A/J mouse. The purpose of this study was to develop new protocols for the isolation and culture of type II and Clara cells from the mouse lung. Both type II and Clara cells were obtained in high purity using a sequential centrifugal elutriation protocol. In the first elutriation, cell fractions were collected using a Standard chamber. The type II and Clara cell fractions were then elutriated separately (two different separations) using a Sanderson chamber. The final purity of the type II and Clara cell preparations was 73% and 76%, respectively. Colonies of 4 to 20 Clara cells exhibiting epithelial morphology were evident 1 wk after plating in low serum medium. The growth of type II cells required the addition of bronchioalveolar lavage fluid and acidic fibroblast growth factor to the medium. The isolation of viable mouse type II and Clara cells in high purity should facilitate the identification of cell-specific changes in gene expressions or in enzymatic pathways following in vivo or in vitro exposure to environmental carcinogens.     

Specificity studies of 3-mercaptopyruvate sulfurtransferase

3-Mercaptopyruvate sulfurtransferase (E.C. 2.8.1.2; MST) is an enzyme believed to function in the endogenous cyanide (CN) detoxification system because it is capable of transferring sulfur from 3-mercaptopyruvate (3-MP) to CN, forming the less toxic thiocyanate (SCN). To date, 3-MP is the only known sulfur-donor substrate for MST. In an effort to increase the understanding of what chemical properties of 3-MP affect its utilization as a substrate, in vitro enzyme kinetic studies of MST were conducted using two mercaptic acids that are structurally related to 3-MP. Neither of these compounds was able to serve as a sulfur-donor substrate for MST. Inhibitor studies determined that 3-mercaptopropionic acid did not affect the K_m of MST for 3-MP but did decrease V_max and, thus, was determined to be a noncompetitive inhibitor. Alternatively, 2-mercaptopropionic acid 2-MPA decreased K_m and V_max and was determined to be an uncompetitive inhibitor of MST with respect to 3-MP. These data indicate that the α-keto group of 3-MP is necessary for its utilization as a substrate, and the inhibitor studies suggest that the position of the sulfur may also affect the binding of these compounds to the enzyme. These observations increase the understanding of what factors can affect the utilization of a compound as a sulfur-donor substrate for MST and may aid in the development of alternative sulfur-donor substrates for MST. © 1996 John Wiley & Sons, Inc.     

Expression and purification strategies for the production of single-chain antibody and T-cell receptor fragments inE. coli

This work describes protocols for the production of single-chain antibody and T-cell receptor fragments inE. coli. A choice of methods is given for the purification of the recombinant fragments that rely on the use of either immunoaffinity or metal chelate affinity chromatography. The TCR fragments may have to be denatured and refolded before the fragments attain their proper conformation.     

Diurnal rhythm of galanin-like immunoreactivity in the paraventricular and suprachiasmatic nuclei and other hypothalamic areas

The peptide galanin (GAL), when injected into the rat hypothalamus, is known to stimulate feeding behavior and affect the secretion of various hormones, including insulin and the adrenal steroid, corticosterone. To determine whether endogenous peptide levels shift in relation to natural rhythms of feeding and circulating hormone levels, rats were sacrificed at different times of the light/dark cycle, and their GAL levels were measured, via radioimmunoassay, in medial hypothalamic dissections and micropunched hypothalamic areas. The results suggest the existence of two distinct diurnal rhythms for hypothalamic GAL. One rhythm, detected exclusively in the area of the SCN, is characterized by bimodal peaks of GAL, threefold higher than basal peptide levels, around the onset of the dark and light periods. The second rhythm shows a single peak of GAL towards the middle of the nocturnal feeding cycle, specifically between the third and sixth hour. This latter rhythm is evident in the dorsal region of the medial hypothalamus, localized specifically to the lateral portion of the PVN. Moreover, it is inversely related to circulating insulin but unrelated to the adrenal steroids, suggesting a possible association between this pancreatic hormone and GAL in the PVN.     

Desulfurization of dibenzothiophene to 2-hydroxybiphenyl by some newly isolated bacterial strains

Gram-positive, non-spore-forming, non-acid-fast, rod-shaped aerobic bacteria with the ability to desulfurize dibenzothiophene (DBT) or dibenzosulfone (DBTO₂) were isolated from soil samples contaminated with fossil fuels. Using a bioavailability method, cells with the desired DbtS⁺ phenotype were enriched. Modified fluorescence and colorimetric assays were used for the initial detection of 2-hydroxybiphenyl (OH-BP) in microtiter plates; subsequently, isolates were grown in wells of microtiter plates and screened for the production of desulfurization product. Fluorescence under UV light and the production of colored product in the phenol assay were used as presumptive indications of production of OH-BP. Confirmation of the presence of OH-BP was achieved with HPLC, UV-absorbance, and mass spectrometry. Nutrient utilization and fatty acid composition (as discerned with Biolog plates and gas chromatography, respectively) were used to identify presumptively the strains as Rhodococcus erythropolis; colony and cell morphology may not be consistent with the identification achieved by nutrient utilization and fatty acid composition. The desulfurization end product, OH-BP, can not be used as carbon source by the tested strain, N1-36.