A screen for genes expressed in Drosophila imaginal discs

The development of Drosophila imaginal discs serves as a model system to understand how genes determine the shape and size of an organ. The identification of genes involved in this process is an important step towards this goal. Here we describe a P-element based enhancer trap screen for genes expressed in the larval imaginal discs. Our aim was to establish a large collection of enhancer trap lines each showing expression of Gal4 in imaginal discs. To this end, we improved the well established P-element vector pGawB in order to obtain higher in vivo transposition frequencies. In addition we chose an F1-screening approach using UAS-GFP as a reporter gene. This system permits the efficient screening of larval and pupal stages of living animals and the detection of imaginal gene expression patterns through the transparent cuticle. The procedure has been optimized for high-throughput. 2'000 P-element insertions have been established which exhibit expression in imaginal discs.     

pGD vectors: versatile tools for the expression of green and red fluorescent protein fusions in agroinfiltrated plant leaves

We have constructed a matched set of binary vectors designated pGD, pGDG and pGDR for the expression and co-localization of native proteins and GFP or DsRed fusions in large numbers of plant cells. The utility of these vectors following agroinfiltration into leaves has been demonstrated with four genes from Sonchus yellow net virus, a plant nucleorhabdovirus, and with a nucleolar marker protein. Of the three SYNV proteins tested, sc4 gave identical localization patterns at the cell wall and nucleus when fused to GFP or DsRed. However, some differences in expression patterns were observed depending on whether DsRed or GFP was the fusion partner. In this regard, the DsRed:P fusion showed a similar pattern of localization to GFP:P, but localized foci appeared in the nucleus and near the periphery of the nucleus. Nevertheless, the viral nucleocapsid protein, expressed as a GFP:N fusion, co-localized with DsRed:P in a subnuclear locale in agreement with our previous observations (Goodin et al., 2001). This locale appears to be distinct from the nucleolus as indicated by co-expression of the N protein, DsRed:P and a nucleolar marker AtFib1 fused to GFP. The SYNV M protein, which is believed to be particularly prone to oligomerization, was detectable only as a GFP fusion. Our results indicate that agroinfiltration with bacteria containing the pGD vectors is extremely useful for transient expression of several proteins in a high proportion of the cells of Nicotiana benthamiana leaves. The GFP and DsRed elements incorporated into the pGD system should greatly increase the ease of visualizing co-localization and interactions of proteins in a variety of experimental dicotyledonous hosts.     

MLL targets SET domain methyltransferase activity to Hox gene promoters

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. We show that MLL regulates target Hox gene expression through direct binding to promoter sequences. We further show that the MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins.     

Effect of atorvastatin on plasma apoE metabolism in patients with combined hyperlipidemia

Atorvastatin, a synthetic HMG-CoA reductase inhibitor used for the treatment of hyperlipidemia and the prevention of coronary artery disease, significantly lowers plasma cholesterol and low-density lipoprotein cholesterol (LDL-C) levels. It also reduces total plasma triglyceride and apoE concentrations. In view of the direct involvement of apoE in the pathogenesis of atherosclerosis, we have investigated the effect of atorvastatin treatment (40 mg/day) on in vivo rates of plasma apoE production and catabolism in six patients with combined hyperlipidemia using a primed constant infusion of deuterated leucine. Atorvastatin treatment resulted in a significant decrease (i.e., 30-37%) in levels of total triglyceride, cholesterol, LDL-C, and apoB in all six patients. Total plasma apoE concentration was reduced from 7.4 +/- 0.9 to 4.3 +/- 0.2 mg/dl (-38 +/- 8%, P < 0.05), predominantly due to a decrease in VLDL apoE (3.4 +/- 0.8 vs. 1.7 +/- 0.2 mg/dl; -42 +/- 11%) and IDL/LDL apoE (1.9 +/- 0.3 vs. 0.8 +/- 0.1 mg/dl; -57 +/- 6%). Total plasma lipoprotein apoE transport (i.e., production) was significantly reduced from 4.67 +/- 0.39 to 3.04 +/- 0.51 mg/kg/day (-34 +/- 10%, P < 0.05) and VLDL apoE transport was reduced from 3.82 +/- 0.67 to 2.26 +/- 0.42 mg/kg/day (-36 +/- 10%, P = 0.057). Plasma and VLDL apoE residence times and HDL apoE kinetic parameters were not significantly affected by drug treatment. Percentage decreases in VLDL apoE concentration and VLDL apoE production were significantly correlated with drug-induced reductions in VLDL triglyceride concentration (r = 0.99, P < 0.001; r = 0.88, P < 0.05, respectively, n = 6). Our results demonstrate that atorvastatin causes a pronounced decrease in total plasma and VLDL apoE concentrations and a significant decrease in plasma and VLDL apoE rates of production in patients with combined hyperlipidemia.     

Relationship between FTC 'tar' and urine mutagenicity in smokers of tobacco-burning or Eclipse cigarettes

The US Federal Trade Commission (FTC) classifies domestic cigarettes into one of three 'tar' categories based on 'tar' and nicotine levels. The objective of the present study was to determine urine mutagenicity in groups of smokers of ultra-low 'tar' (ULT), full-flavor low 'tar' (FFLT) and full-flavor 'tar' (FF) filtered cigarettes after switching to primarily tobacco-heating Eclipse cigarettes. Sixty-seven smokers maintained a specified diet and consumed ad libitum their usual brands of cigarettes, switched to Eclipse, and switched back to their usual brands. Twenty-four hour urine samples were collected weekly, concentrated on XAD-2 resin, and tested in the Ames mutagenicity assay using bacterial strains TA98 and YG1024 with S9 metabolic activation. Daily consumption of cigarettes was not significantly different (at P<0.05) between FTC 'tar' categories and average daily cigarette consumption did not change significantly in any smoker group after switching to Eclipse cigarettes. Average urine mutagenicity was 47% less (P<0.05) for ULT than for FFLT usual brand smokers as measured by the more sensitive strain YG1024, although no significant differences (P<0.05) were observed in urine mutagenicity between usual brand FTC 'tar' categories as measured by strain TA98. The reduction in urinary mutagens in the more sensitive strain, YG1024, observed in ULT smokers as compared with higher 'tar' categories suggest reduced exposure to mutagens. Usual brand salivary cotinine in the ULT group was significantly lower (P<0.05) than the FF group and the FFLT group. Salivary cotinine did not differ significantly (at P<0.05) among the smoker groups when smoking Eclipse compared to usual brand. After switching to Eclipse, the following reductions in urinary mutagenicity were observed: ULT, 70.1+/-6.4% (TA98), 70.9+/-6.2% (YG1024); FFLT, 77.1+/-2.4% (TA98), 73.6+/-2.0% (YG1024); and FF, 76.1+/-3.5% (TA98), 71.4+/-4.0% (YG1024). Across all 'tar' categories, cigarette smokers experienced significant reductions (P<0.05) in urine mutagenicity, but not salivary cotinine, upon switching to Eclipse. The reduction in urine mutagenicity when smoking Eclipse provides supporting evidence that Eclipse may present less risk of cancer compared to cigarettes currently in the market.     

Ingestion of plant secondary compounds causes diuresis in desert herbivores

Plant secondary compounds are recognized deterrents and toxins to a variety of herbivores. The effect of secondary compounds on water balance of herbivores is virtually unexplored, yet secondary compounds could potentially cause a decrease in an animal's ability to maintain water balance. We investigated the effects of secondary compounds, alpha-pinene and creosote resin, on water balance in three species of herbivorous woodrats (Neotoma stephensi, N. albigula, N. lepida). In separate experiments, we measured the effect of these secondary compounds on voluntary water consumption, urine volume and urine osmolarity. In both experiments, water intake and urine volume increased and urine osmolarity decreased compared to controls. Water balance of specialist or experienced woodrats was less affected than generalists and woodrats with less prior experience with particular secondary compounds. Our results suggest that secondary compounds have diuretic-like effects on herbivores. Woodrats live in arid habitats with limited access to freestanding water; thus an increase in water requirements may have profound consequences on foraging behavior and fitness.     

The pam1 gene is required for meiotic bouquet formation and efficient homologous synapsis in maize (Zea mays L.)

The clustering of telomeres on the nuclear envelope (NE) during meiotic prophase to form the bouquet arrangement of chromosomes may facilitate homologous chromosome synapsis. The pam1 (plural abnormalities of meiosis 1) gene is the first maize gene that appears to be required for telomere clustering, and homologous synapsis is impaired in pam1. Telomere clustering on the NE is arrested or delayed at an intermediate stage in pam1. Telomeres associate with the NE during the leptotene-zygotene transition but cluster slowly if at all as meiosis proceeds. Intermediate stages in telomere clustering including miniclusters are observed in pam1 but not in wild-type meiocytes. The tight bouquet normally seen at zygotene is a rare event. In contrast, the polarization of centromeres vs. telomeres in the nucleus at the leptotene-zygotene transition is the same in mutant and wild-type cells. Defects in homologous chromosome synapsis include incomplete synapsis, nonhomologous synapsis, and unresolved interlocks. However, the number of RAD51 foci on chromosomes in pam1 is similar to that of wild type. We suggest that the defects in homologous synapsis and the retardation of prophase I arise from the irregularity of telomere clustering and propose that pam1 is involved in the control of bouquet formation and downstream meiotic prophase I events.