The tomato homolog of the gene encoding UV-damaged DNA binding protein 1 (DDB1) underlined as the gene that causes the<Emphasis Type="Italic"> high pigment-1</Emphasis> mutant phenotype

A tomato EST sequence, highly homologous to the human and Arabidopsis thaliana UV-damaged DNA binding protein 1 (DDB1), was mapped to the centromeric region of the tomato chromosome 2. This region was previously shown to harbor the HP-1 gene, encoding the high pigment-1 (hp-1) and the high pigment-1^w (hp-1^w) mutant phenotypes. Recent results also show that the A. thaliana DDB1 protein interacts both genetically and biochemically with the protein encoded by DEETIOLATED1, a gene carrying three tomato mutations that are in many respects isophenotypic to hp-1: high pigment-2 (hp-2), high pigment-2^j (hp-2^j) and dark green (dg). The entire coding region of the DDB1 gene was sequenced in an hp-1 mutant and its near-isogenic normal plant in the cv. Ailsa Craig background, and also in an hp-1^w mutant and its isogenic normal plant in the GT breeding line background. Sequence analysis revealed a single A⁹³¹-to-T⁹³¹ base transversion in the coding sequence of the DDB1 gene in the hp-1 mutant plants. This transversion results in the substitution of the conserved asparagine at position 311 to a tyrosine residue. In the hp-1^w mutant, on the other hand, a single G²³⁹²-to-A²³⁹² transition was observed, resulting in the substitution of the conserved glutamic acid at position 798 to a lysine residue. The single nucleotide polymorphism that differentiates hp-1 mutant and normal plants in the cv. Ailsa Craig background was used to design a pyrosequencing genotyping system. Analysis of a resource F₂ population segregating for the hp-1 mutation revealed a very strong linkage association between the DDB1 locus and the photomorphogenic response of the seedlings, measured as hypocotyl length (25<LOD score<26, R²=62.8%). These results strongly support the hypothesis that DDB1 is the gene encoding the hp-1 and hp-1^w mutant phenotypes.Communicated by R. Hagemann     

The presence of both growth inhibitory and growth stimulatory factors on membranes prepared from mouse liver

Plasma membrane fractions from mouse livers were examined for the presence of growth regulatory peptides. Both growth inhibitory and growth stimulatory factors were found on these membranes. The growth inhibitory component could be enriched by extractions with both dimethylmaleic anhydride and octylglucoside. The growth stimulatory component could be removed from the membrane by either freeze-thaw, high salt, protease or pyrophosphate treatment, indicating that this factor is an extrinsic membrane protein. The existence of these factors on liver membranes provides an easily obtainable source for the large-scale purification of these molecules and may indicate a possible role in normal tissue growth.     

Genetic variation in C57BL/6 ES cell lines and genetic instability in the Bruce4 C57BL/6 ES cell line

Genetically modified mouse strains derived from embryonic stem (ES) cells are powerful tools for gene function analysis. ES cells from the C57BL/6 mouse strain are not widely used to generate mouse models despite the advantage of a defined genetic background. We assessed genetic variation in six such ES cell lines with 275 SSLP markers. Compared to C57BL/6, Bruce4 differed at 34 SSLP markers and had significant heterozygosity on three chromosomes. BL/6#3 and Dale1 ES cell lines differed at only 3 SSLP makers. The C2 and WB6d ES cell lines differed at 6 SSLP markers. It is important to compare the efficiency of producing mouse models with available C57BL/6 ES cells relative to standard 129 mouse strain ES cells. We assessed genetic stability (the tendency of cells to become aneuploid) in 110 gene-targeted ES cell clones from the most widely used C57BL/6 ES cell line, Bruce4, and 710 targeted 129 ES cell clones. Bruce4 clones were more likely to be aneuploid and unsuitable for ES cell-mouse chimera production. Despite their tendency to aneuploidy and consequent inefficiency, use of Bruce4 ES cells can be valuable for models requiring behavioral studies and other mouse models that benefit from a defined C57BL/6 background. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Branched-chain amino acid supplementation and human performance when hypohydrated in the heat.

The serotonin system may contribute to reduced human performance when hypohydrated in the heat. This study determined whether branched-chain amino acid (BCAA) supplementation could sustain exercise and cognitive performance in the heat (40 degrees C dry bulb, 20% relative humidity) when hypohydrated by 4% of body mass. Seven heat-acclimated men completed two experimental trials, each consisting of one preparation and one test day. On day 1, a low-carbohydrate diet was eaten and subjects performed exhaustive cycling (morning) and treadmill exercise in the heat (afternoon) to lower muscle glycogen and achieve the desired hypohydration level. On day 2, subjects consumed an isocaloric BCAA and carbohydrate (BC) or carbohydrate-only drink during exercise. Experimental trials included 60 min of cycle ergometry (50% peak oxygen uptake) followed by a 30-min time trial in the heat. A cognitive test battery was completed before and after exercise, and blood samples were taken. BC produced a 2.5-fold increase (P < 0.05) in plasma BCAA and lowered (P < 0.05) the ratios of total tryptophan to BCAA and large neutral amino acid. Blood prolactin, glucose, lactate, and osmolality were not different between trials but increased over time. Cardiovascular and thermoregulatory data were also similar between trials. BC did not alter time-trial performance, cognitive performance, mood, perceived exertion, or perceived thermal comfort. We conclude that BCAA does not alter exercise or cognitive performance in the heat when subjects are hypohydrated.     

Central role of double-stranded RNA-activated protein kinase in microbial induction of nitric oxide synthase

NO synthase 2 (NOS2) is induced in airway epithelium by influenza virus infection. NOS2 induction late in the course of viral infection may occur in response to IFN-gamma, but early in infection gene expression may be induced by the viral replicative intermediate dsRNA through the dsRNA-activated protein kinase (PKR). Since PKR activates signaling pathways important in NOS2 gene induction, we determined whether PKR is a component in the signal transduction pathway leading to NOS2 gene expression after viral infection of airway epithelium. We show that NOS2 gene expression in human airway epithelial cells occurs in response to influenza A virus or synthetic dsRNA. Furthermore, dsRNA leads to rapid activation of PKR, followed by activation of signaling components including NF-kappaB and IFN regulatory factor 1. NOS2 expression is markedly diminished and IFN regulatory factor 1 and NF-kappaB activation are substantially impaired in PKR null cells. Strikingly, NOS2 induction in response to LPS is abolished in PKR null cells, confirming a central role for PKR in the general signaling pathway to NOS2.