Utility of different gene enrichment approaches toward identifying and sequencing the maize gene space

Maize (Zea mays) possesses a large, highly repetitive genome, and subsequently a number of reduced-representation sequencing approaches have been used to try and enrich for gene space while eluding difficulties associated with repetitive DNA. This article documents the ability of publicly available maize expressed sequence tag and Genome Survey Sequences (GSSs; many of which were isolated through the use of reduced representation techniques) to recognize and provide coverage of 78 maize full-length cDNAs (FLCs). All 78 FLCs in the dataset were identified by at least three GSSs, indicating that the majority of maize genes have been identified by at least one currently available GSS. Both methyl-filtration and high-Cot enrichment methods provided a 7- to 8-fold increase in gene discovery rates as compared to random sequencing. The available maize GSSs aligned to 75% of the FLC nucleotides used to perform searches, while the expressed sequence tag sequences aligned to 73% of the nucleotides. Our data suggest that at least approximately 95% of maize genes have been tagged by at least one GSS. While the GSSs are very effective for gene identification, relatively few (18%) of the FLCs are completely represented by GSSs. Analysis of the overlap of coverage and bias due to position within a gene suggest that RescueMu, methyl-filtration, and high-Cot methods are at least partially nonredundant.     

Regulation of protein kinases; controlling activity through activation segment conformation

There are currently at least forty-six unique protein kinase crystal structures, twenty-four of which are available in an active state. Here we examine these structures using a structural bioinformatics approach to understand how the conformation of the activation segment controls kinase activity.     

Predicting function from structure: 3D structure studies of the mammalian Golgi complex

3D electron tomography studies of the structure of the mammalian Golgi complex have led to four functional predictions (1). The sorting and exit site from the Golgi comprises two or three distinct trans-cisternae (2). The docking of vesicular-tubular clusters at the cis-face and the fragmentation of trans-cisternae are coordinated (3). The mechanisms of transport through, and exit from, the Golgi vary with physiological state, and in different cells and tissues (4). Specialized trans-ER functions in the delivery of ceramide to sphingomyelin synthase in the trans-Golgi membrane, for the regulated sorting via sphingolipid-cholesterol-rich domains. These structure-based predictions can now be tested using a variety of powerful cell and molecular tools.     

Modeling Sustainability of Arctic Communities: An Interdisciplinary Collaboration of Researchers and Local Knowledge Holders

How will climate change affect the sustainability of Arctic villages over the next 40 years? This question motivated a collaboration of 23 researchers and four Arctic communities (Old Crow, Yukon Territory, Canada; Aklavik, Northwest Territories, Canada; Fort McPherson, Northwest Territories, Canada; and Arctic Village, Alaska, USA) in or near the range of the Porcupine Caribou Herd. We drew on existing research and local knowledge to examine potential effects of climate change, petroleum development, tourism, and government spending cutbacks on the sustainability of four Arctic villages. We used data across eight disciplines to develop an Arctic Community Synthesis Model and a Web-based, interactive Possible Futures Model. Results suggested that climate warming will increase vegetation biomass within the herd’s summer range. However, despite forage increasing, the herd was projected as likely to decline with a warming climate because of increased insect harassment in the summer and potentially greater winter snow depths. There was a strong negative correlation between hypothetical, development-induced displacement of cows and calves from utilized calving grounds and calf survival during June. The results suggested that climate warming coupled with petroleum development would cause a decline in caribou harvest by local communities. Because the Synthesis Model inherits uncertainties associated with each component model, sensitivity analysis is required. Scientists and stakeholders agreed that (1) although simulation models are incomplete abstractions of the real world, they helped bring scientific and community knowledge together, and (2) relationships established across disciplines and between scientists and communities were a valuable outcome of the study. Additional project materials, including the Web-based Possible Futures Model, are available at http://www.taiga.net/sustain.     

The Effects of Infrared Loading and Water Table on Soil Energy Fluxes in Northern Peatlands

Increased radiative forcing is an inevitable part of global climate change, yet little is known of its potential effects on the energy fluxes in natural ecosystems. To simulate the conditions of global warming, we exposed peat monoliths (depth, 0.6 m; surface area, 2.1 m²) from a bog and fen in northern Minnesota, USA, to three infrared (IR) loading (ambient, +45, and +90 W m^–2) and three water table (–16, –20, and –29 cm in bog and –1, –10 and –18 cm in fen) treatments, each replicated in three mesocosm plots. Net radiation (Rn) and soil energy fluxes at the top, bottom, and sides of the mesocosms were measured in 1999, 5 years after the treatments had begun. Soil heat flux (G) increased proportionately with IR loading, comprising about 3%–8% of Rn. In the fen, the effect of IR loading on G was modulated by water table depth, whereas in the bog it was not. Energy dissipation from the mesocosms occurred mainly via vertical exchange with air, as well as with deeper soil layers through the bottom of the mesocosms, whereas lateral fluxes were 10–20-fold smaller and independent of IR loading and water table depth. The exchange with deeper soil layers was sensitive to water table depth, in contrast to G, which responded primarily to IR loading. The qualitative responses in the bog and fen were similar, but the fen displayed wider seasonal variation and greater extremes in soil energy fluxes. The differences of G in the bog and fen are attributed to differences in the reflectance in the long waveband as a function of vegetation type, whereas the differences in soil heat storage may also depend on different soil properties and different water table depth at comparable treatments. These data suggest that the ecosystem-dependent controls over soil energy fluxes may provide an important constraint on biotic response to climate change.     

Characterization of the 70S Ribosome from Rhodopseudomonas palustris using an integrated "top-down" and "bottom-up" mass spectrometric approach

We present a comprehensive mass spectrometric approach that integrates intact protein molecular mass measurement ("top-down") and proteolytic fragment identification ("bottom-up") to characterize the 70S ribosome from Rhodopseudomonas palustris. Forty-two intact protein identifications were obtained by the top-down approach and 53 out of the 54 orthologs to Escherichia coli ribosomal proteins were identified from bottom-up analysis. This integrated approach simplified the assignment of post-translational modifications by increasing the confidence of identifications, distinguishing between isoforms, and identifying the amino acid positions at which particular post-translational modifications occurred. Our combined mass spectrometry data also allowed us to check and validate the gene annotations for three ribosomal proteins predicted to possess extended C-termini. In particular, we identified a highly repetitive C-terminal "alanine tail" on L25. This type of low complexity sequence, common to eukaryotic proteins, has previously not been reported in prokaryotic proteins. To our knowledge, this is the most comprehensive protein complex analysis to date that integrates two MS techniques.     

Arabidopsis thaliana GLN2-encoded glutamine synthetase is dual targeted to leaf mitochondria and chloroplasts

In higher plants, photorespiratory Gly oxidation in leaf mitochondria yields ammonium in large amounts. Mitochondrial ammonium must somehow be recovered as glutamate in chloroplasts. As the first step in that recovery, we report glutamine synthetase (GS) activity in highly purified Arabidopsis thaliana mitochondria isolated from light-adapted leaf tissue. Leaf mitochondrial GS activity is further induced in response to either physiological CO(2) limitation or transient darkness. Historically, whether mitochondria are fully competent for oxidative phosphorylation in actively photorespiring leaves has remained uncertain. Here, we report that light-adapted, intact, leaf mitochondria supplied with Gly as sole energy source are fully competent for oxidative phosphorylation. Purified intact mitochondria efficiently use Gly oxidation (as sole energy, NH(3), and CO(2) source) to drive conversion of l-Orn to l-citrulline, an ATP-dependent process. An A. thaliana genome-wide search for nuclear gene(s) encoding mitochondrial GS activity yielded a single candidate, GLN2. Stably transgenic A. thaliana ecotype Columbia plants expressing a p35S::GLN2::green fluorescent protein (GFP) chimeric reporter were constructed. When observed by laser scanning confocal microscopy, leaf mesophyll and epidermal tissue of transgenic plants showed punctate GFP fluorescence that colocalized with mitochondria. In immunoblot experiments, a 41-kD chimeric GLN2::GFP protein was present in both leaf mitochondria and chloroplasts of these stably transgenic plants. Therefore, the GLN2 gene product, heretofore labeled plastidic GS-2, functions in both leaf mitochondria and chloroplasts to faciliate ammonium recovery during photorespiration.     

Effects of aging on capillary geometry and hemodynamics in rat spinotrapezius muscle

The effects of aging on muscle microvascular structure and function may play a key role in performance deficits and impairment of O2 exchange within skeletal muscle of senescent individuals. To determine the effects of aging on capillary geometry, red blood cell (RBC) hemodynamics, and hematocrit in a muscle of mixed fiber type, spinotrapezius muscles from Fischer 344 x Brown Norway hybrid rats aged 6-8 mo [young (Y); body mass 421 +/- 10 g, n = 6] and 26-28 mo [old (O); 561 +/- 12 g, n = 6] were observed by high-resolution transmission light microscopy under resting conditions. The percentage of RBC-perfused capillaries (Y: 78 +/- 3%; O: 75 +/- 2%) and degree of tortuosity and branching (Y: 13 +/- 2%; O: 13 +/- 2%, additional capillary length) were not different in O vs. Y muscles. Lineal density of RBC-perfused capillaries in O was significantly reduced (Y: 30.7 +/- 1.8, O: 22.8 +/- 3.1 capillaries/mm; P < 0.05). However, RBC-perfused capillaries from O rats (n = 78) exhibited increased RBC velocity (VRBC) (Y: 219 +/- 12, O: 310 +/- 14 microm/s; P < 0.05) and RBC flux (FRBC) (Y: 27 +/- 2, O: 41 +/- 2 RBC/s; P < 0.05) vs. Y rats (n = 66). Thus O2 delivery per unit of muscle was not different between groups (Y: 894 +/- 111, O: 887 +/- 118 RBC. s-1. mm muscle-1). Capillary hematocrit was not different in Y vs. O rats (Y: 26 +/- 1%, O: 28 +/- 1%: P > 0.05). These data indicate that in resting spinotrapezius muscle, aging decreases the lineal density of RBC-perfused capillaries while increasing mean VRBC and FRBC within those capillaries. Whereas muscle conductive O2 delivery and capillary hematocrit were unchanged, elevated VRBC reduces capillary RBC transit time and may impair the diffusive transport of O2 from blood to myocyte particularly under exercise conditions.     

Nitric oxide and attenuated reflex cutaneous vasodilation in aged skin

Thermoregulatory cutaneous vasodilation is diminished in the elderly. The goal of this study was to test the hypothesis that a reduction in nitric oxide (NO)-dependent mechanisms contributes to the attenuated reflex cutaneous vasodilation in older subjects. Seven young (23 +/- 2 yr) and seven older (71 +/- 6 yr) men were instrumented with two microdialysis fibers in the forearm skin. One site served as control (Ringer infusion), and the second site was perfused with 10 mM N(G)-nitro-l-arginine methyl ester to inhibit NO synthase (NOS) throughout the protocol. Water-perfused suits were used to raise core temperature 1.0 degrees C. Red blood cell (RBC) flux was measured with laser-Doppler flowmetry over each microdialysis fiber. Cutaneous vascular conductance (CVC) was calculated as RBC flux per mean arterial pressure, with values expressed as a percentage of maximal vasodilation (infusion of 28 mM sodium nitroprusside). NOS inhibition reduced CVC from 75 +/- 6% maximal CVC (CVC(max)) to 53 +/- 3% CVC(max) in the young subjects and from 64 +/- 5% CVC(max) to 29 +/- 2% CVC(max) in the older subjects with a 1.0 degrees C rise in core temperature. Thus the relative NO-dependent portion of cutaneous active vasodilation (AVD) accounted for approximately 23% of vasodilation in the young subjects and 60% of the vasodilation in the older subjects at this level of hyperthermia (P < 0.001). In summary, NO-mediated pathways contributed more to the total vasodilatory response of the older subjects at high core temperatures. This suggests that attenuated cutaneous vasodilation with age may be due to a reduction in, or decreased vascular responsiveness to, the unknown neurotransmitter(s) mediating AVD.