A viral suppressor of gene silencing in plants

Gene silencing is an important but little understood regulatory mechanism in plants. Here we report that a viral sequence, initially identified as a mediator of synergistic viral disease, acts to suppress the establishment of both transgene-induced and virus-induced posttranscriptional gene silencing. The viral suppressor of silencing comprises the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, helper component-proteinase (HC-Pro) and a small part of P3, and is termed the P1/HC-Pro sequence. A reversal of silencing assay was used to assess the effect of the P1/HC-Pro sequence on transgenic tobacco plants (line T4) that are posttranscriptionally silenced for the uidA reporter gene. Silencing was lifted in offspring of T4 crosses with four independent transgenic lines expressing P1/HC-Pro, but not in offspring of control crosses. Viral vectors were used to assess the effect of P1/HC-Pro expression on virus-induced gene silencing (VIGS). The ability of a potato virus X vector expressing green fluorescent protein to induce silencing of a green fluorescent protein transgene was eliminated or greatly reduced when P1/HC-Pro was expressed from the same vector or from coinfecting potato virus X vectors. Expression of the HC-Pro coding sequence alone was sufficient to suppress virus-induced gene silencing, and the HC-Pro protein product was required for the suppression. This discovery points to the role of gene silencing as a natural antiviral defense system in plants and offers different approaches to elucidate the molecular basis of gene silencing.

Disruption of the murine gene encoding phosphatidylethanolamine N-methyltransferase

All nucleated cells make phosphatidylcholine via the CDP-choline pathway. Liver has an alternative pathway in which phosphatidylcholine is made by methylation of phosphatidylethanolamine catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). We investigated the function of PEMT and its role in animal physiology by targeted disruption of its gene, Pempt2. A targeting vector that interrupts exon 2 was constructed and introduced into mice yielding three genotypes: normal (+/+), heterozygotes (+/−), and homozygotes (−/−) for the disrupted PEMT gene. Only a trace of PE methylation activity remained in Pempt2(−/−) mice. Antibody to one form of the enzyme, PEMT2, indicated complete loss of this protein from Pempt2(−/−) mice and a decrease in Pempt2(+/−) mice, compared with Pempt2(+/+) mice. The levels of hepatic phosphatidylethanolamine and phosphatidylcholine were minimally affected. The active form of CTP:phosphocholine cytidylyltransferase, the regulated enzyme in the CDP-choline pathway, was increased 60% in the PEMT-deficient mice. Injection of [l-methyl-3H]methionine demonstrated that the in vivo PEMT activity was eliminated in the Pempt2(−/−) mice and markedly decreased in the Pempt2(+/−) mice. This experiment also demonstrated that the choline moiety derived from PEMT in the liver can be distributed via the plasma throughout the mouse where it is found as phosphatidylcholine, lysophosphatidylcholine, and sphingomyelin. Mice homozygous for the disrupted Pempt2 gene displayed no abnormal phenotype, normal hepatocyte morphology, normal plasma lipid levels and no differences in bile composition. This is the first application of the “knockout mouse” technique to a gene for phospholipid biosynthesis.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Genetic Regulation and Cellular Expression1

NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14) is a key enzyme in primary nitrogen assimilation in alfalfa (Medicago sativa L.) root nodules. Here we report that in alfalfa, a single gene, probably with multiple alleles, encodes for NADH-GOGAT. In situ hybridizations were performed to assess the location of NADH-GOGAT transcript in alfalfa root nodules. In wild-type cv Saranac nodules the NADH-GOGAT gene is predominantly expressed in infected cells. Nodules devoid of bacteroids (empty) induced by Sinorhizobium meliloti 7154 had no NADH-GOGAT transcript detectable by in situ hybridization, suggesting that the presence of the bacteroid may be important for NADH-GOGAT expression. The pattern of expression of NADH-GOGAT shifted during root nodule development. Until d 9 after planting, all infected cells appeared to express NADH-GOGAT. By d 19, a gradient of expression from high in the early symbiotic zone to low in the late symbiotic zone was observed. In 33-d-old nodules expression was seen in only a few cell layers in the early symbiotic zone. This pattern of expression was also observed for the nifH transcript but not for leghemoglobin. The promoter of NADH-GOGAT was evaluated in transgenic alfalfa plants carrying chimeric β-glucuronidase promoter fusions. The results suggest that there are at least four regulatory elements. The region responsible for expression in the infected cell zone contains an 88-bp direct repeat.

NADH-Glutamate Synthase in Alfalfa Root Nodules. Immunocytochemical Localization1

In root nodules of alfalfa (Medicago sativa L.), N2 is reduced to NH4+ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH4+ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N2.

Discovering a history : the school of art at the University of Denver

Discovering a History: The School of Art at the University of Denver explores the early history of art education in Denver, and the significance of visual art education at the University of Denver within that history beginning in 1865, when the first classes in art were offered, and ending in 1929 when the University acquired the Chappell School of Art—an independent art school—and appointed Vance Kirkland as director. This paper also explores competing art institutions, which at times posed great hindrances to the University. Further, it illustrates how the artists who taught at the University of Denver School of Art, such as Ida De Steiguer, Preston Powers, Emma Richardson Cherry, and Henry Read, were amongst the great contributors to Denver’s burgeoning artistic culture.

Massachusetts, Connecticut, and Rhode Island, 1825 (Raster Image)

This layer is a georeferenced raster image of the historic paper map entitled: Map of Massachusetts, Connecticut and Rhode Island : constructed from the latest authorities, drawn by D.H. Vance ; engraved by J.H. Young. It was published by A. Finley in 1825. Scale [ca. 1:700,000]. Covers also portions of New York, New Jersey, Vermont, and New Hampshire. The image inside the map neatline is georeferenced to the surface of the earth and fit to the USA Contiguous Albers Equal Area Conic projection (Meters). All map collar and inset information is also available as part of the raster image, including any inset maps, profiles, statistical tables, directories, text, illustrations, or other information associated with the principal map. This map shows features such as roads, drainage, state, county, and town boundaries, and more. Relief is shown pictorially. Includes statistical table. This layer is part of a selection of digitally scanned and georeferenced historic maps of New England from the Harvard Map Collection. These maps typically portray both natural and manmade features. The selection represents a range of regions, originators, ground condition dates, scales, and purposes.

Sm-Nd isotope and Mn/Ca ratios for cleaned Neogloboquadrina pachyderma from ODP Hole 105-647A (Table 1)

The neodymium (Nd) isotope composition of ancient seawater is a potentially useful tracer of changes in continental inputs and ocean circulation on timescales of a few ka. Here we present the first Nd isotope record for seawater using sedimentary foraminifera cleaned using standard oxidative-reductive techniques. The data, along with Mn/Ca ratios, suggest that cleaned foraminifera provide a reliable record of Nd in seawater and hold out the prospect of using Nd in foraminifera to examine changes in seawater that accompany glacial-interglacial climatic cycles. The principal potential problem to be overcome with the use of forams as records of trace elements in ancient seawater is their diagenetic Fe-Mn coatings. These contain large amounts of Nd and other trace elements but can be cleaned off using highly reducing reagents. Mn(Ca ratios for the majority of the cleaned sedimentary foraminifera analysed here lie within the range (10-100 µmol/mol) that has yielded success in studies of transition elements in forams. Mass-balance modelling suggests that for residual Mn/Ca ratios <100 µmol/mol, Nd added to the foram in the coating will never shift the measured Nd isotope composition significantly away from the seawater value acquired by the foram test in the water column. Additionally, Nd concentrations measured in cleaned sedimentary foraminifera are comparable with those for a modern sample that has never encountered diagenetic fluids. Finally, core-top planktonic foraminifera for two sites have Nd isotope compositions that are identical to local surface seawater. The data we present here for Labrador Sea forams over the past 2.5 m.y. are interpreted in terms of changes in the seawater isotopic composition. The data show a pronounced shift from epsilon-Nd values of ~-12 to ~-19 in the period 2.5-1.5 Ma. This change is interpreted to result from the initiation of Northern Hemisphere glaciation and the increased derivation of Labrador Sea Nd via ice-rafting from Archaean terranes in central Canada. In combination with stable isotope and foraminiferal relative species abundance data, the new Nd data are consistent with the surface hydrography of the Labrador Sea being dominated by a fluctuating balance between cold, polar waters containing unradiogenic Nd and warm, subtropical waters containing more radiogenic Nd. The major change in Labrador Sea Nd that is observed in the past 2.5 Ma can, on its own, account for the change in the Nd isotope composition of North Atlantic Deep Water over the same time period.