Nascent polypeptide-associated complex protein prevents mistargeting of nascent chains to the endoplasmic reticulum.

We show that, after removal of the nascent polypeptide-associated complex (NAC) from ribosome-associated nascent chains, ribosomes synthesizing proteins lacking signal peptides are efficiently targeted to the endoplasmic reticulum (ER) membrane. After this mistargeting, translocation across the ER membrane occurs, albeit less efficiently than for a nascent secretory polypeptide, perhaps because the signal peptide is needed to catalyze the opening of the translocation pore. The mistargeting was prevented by the addition of purified NAC and was shown not to be mediated by the signal recognition particle and its receptor. Instead, it appears to be a consequence of the intrinsic affinity of ribosomes for membrane binding sites, since it can be blocked by competing ribosomes that lack associated nascent polypeptides. We propose that, when bound to a signalless ribosome-associated nascent polypeptide, NAC sterically blocks the site in the ribosome for membrane binding.

Low major histocompatibility complex class II diversity in European and North American moose.

Major histocompatibility complex (MHC) genes encode cell surface proteins whose function is to bind and present intracellularly processed peptides to T lymphocytes of the immune system. Extensive MHC diversity has been documented in many species and is maintained by some form of balancing selection. We report here that both European and North American populations of moose (Alces alces) exhibit very low levels of genetic diversity at an expressed MHC class II DRB locus. The observed polymorphism was restricted to six amino acid substitutions, all in the peptide binding site, and four of these were shared between continents. The data imply that the moose have lost MHC diversity in a population bottleneck, prior to the divergence of the Old and New World subspecies. Sequence analysis of mtDNA showed that the two subspecies diverged at least 100,000 years ago. Thus, viable moose populations with very restricted MHC diversity have been maintained for a long period of time. Both positive selection for polymorphism and intraexonic recombination have contributed to the generation of MHC diversity after the putative bottleneck.

Dual transcriptional control by Ear3/COUP: negative regulation through the DR1 direct repeat and positive regulation through a sequence downstream of the transcriptional start site of the mouse mammary tumor virus promoter.

Ear3/COUP is an orphan member of the steroid/thyroid hormone receptor superfamily of transcription factors and binds most tightly to a direct repeat of AGGTCA with 1 nucleotide in between (DR1). Ear3/COUP also binds with a similar affinity to the palindromic thyroid hormone response element (TRE). This binding preference of Ear3/COUP is same as that of the retinoid X receptor (RXR), which is another member of the superfamily. In the present study, we identified a sequence responsible for Ear3/COUP-mediated transactivation in the region downstream of the transcription start site of the mouse mammary tumor virus promoter. This cis-acting sequence was unresponsive to RXR. When the DR1 or TRE sequence was added upstream of the promoter, transactivation by Ear3/COUP was completely abolished, whereas RXR enhanced transcription from the promoter. The mode of action of Ear3/COUP could be utilized to control complex gene expressions in morphogenesis, homeostasis, and development.

Mutational analysis of the conserved region 2 site of adenovirus E1A and its effect on binding to the retinoblastoma gene product: use of the "double-tagging" assay.

We have explored the feasibility of using a "double-tagging" assay for assessing which amino acids of a protein are responsible for its binding to another protein. We have chosen the adenovirus E1A-retinoblastoma gene product (pRB) proteins for a model system, and we focused on the high-affinity conserved region 2 of adenovirus E1A (CR2). We used site-specific mutagenesis to generate a mutant E1A gene with a lysine instead of an aspartic acid at position 121 within the CR2 site. We demonstrated that this mutant exhibited little binding to pRB by the double-tagging assay. We also have shown that this lack of binding is not due to any significant decrease in the level of expression of the beta-galactosidase-E1A fusion protein. We then created a "library" of phage expressing beta-galactosidase-E1A fusion proteins with a variety of different mutations within CR2. This library of E1A mutations was used in a double-tagging screening to identify mutant clones that bound to pRB. Three classes of phage were identified: the vast majority of clones were negative and exhibited no binding to pRB. Approximately 1 in 10,000 bound to pRB but not to E1A ("true positives"). A variable number of clones appeared to bind equally well to both pRB and E1A ("false positives"). The DNA sequence of 10 true positive clones yielded the following consensus sequence: DLTCXEX, where X = any amino acid. The recovery of positive clones with only one of several allowed amino acids at each position suggests that most, if not all, of the conserved residues play an important role in binding to pRB. On the other hand, the DNA sequence of the negative clones appeared random. These results are consistent with those obtained from other sources. These data suggest that a double-tagging assay can be employed for determining which amino acids of a protein are important for specifying its interaction with another protein if the complex forms within bacteria. This assay is rapid and up to 1 x 10(6) mutations can be screened at one time.

Modulation of 5' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.

Breaking down complex saproxylic communities: understanding sub-networks structure and implications to network robustness

Saproxylic insect communities inhabiting tree hollow microhabitats correspond with large food webs which simultaneously are constituted by multiple types of plant-animal and animal-animal interactions, according to the use of trophic resources (wood- and insect-dependent sub-networks), or to trophic habits or interaction types (xylophagous, saprophagous, xylomycetophagous, predators and commensals). We quantitatively assessed which properties of specialised networks were present in a complex networks involving different interacting types such as saproxylic community, and how they can be organised in trophic food webs. The architecture, interacting patterns and food web composition were evaluated along sub-networks, analysing their implications to network robustness from random and directed extinction simulations. A structure of large and cohesive modules with weakly connected nodes was observed throughout saproxylic sub-networks, composing the main food webs constituting this community. Insect-dependent sub-networks were more modular than wood-dependent sub-networks. Wood-dependent sub-networks presented higher species degree, connectance, links, linkage density, interaction strength, and were less specialised and more aggregated than insect-dependent sub-networks. These attributes defined high network robustness in wood-dependent sub-networks. Finally, our results emphasise the relevance of modularity, differences among interacting types and interrelations among them in modelling the structure of saproxylic communities and in determining their stability.

(Table T1) Permeability, electrical resistivity and density of ODP Hole 193-1188A and Site 193-1189 minicores, PACMANUS field

Magmatic fluids, heat fluxes, and fluid/rock interactions associated with hydrothermal systems along spreading centers and convergent margins have a significant impact on the genesis of major sulfide deposits and biological communities. Circulation of hydrothermal fluids is one of the most fundamental processes associated with localized mineralization and is controlled by inherent porous and permeable properties of the ocean crust. Heat from magmatic intrusions drives circulation of seawater through permeable portions of the oceanic crust and upper mantle, discharging at the seafloor as both focused high-temperature (250°-400°C) fluids and diffuse lower-temperature (<250°C) fluids. This complex interaction between the circulating hydrothermal fluids and the oceanic basement greatly influences the physical properties and the composition of the crust (Thompson, 1983; Jacobson, 1992, doi:10.1029/91RG02811; Johnson and Semyan, 1994, doi:10.1029/93JB00717). During Ocean Drilling Program (ODP) Leg 193, 13 holes were drilled in the PACMANUS hydrothermal system (Binns, Barriga, Miller, et al., 2002, doi:10.2973/odp.proc.ir.193.2002). The hydrothermal system consists of isolated hydrothermal deposits lined along the main crest of the Pual Ridge, a 500- to 700-m-high felsic neovolcanic ridge in the eastern Manus Basin. The principal drilling targets were the Snowcap (Site 1188) and Roman Ruins (Site 1189) active hydrothermal fields. Samples from these two sites were used for a series of permeability, electrical resistivity, and X-ray computed tomography measurements.

Uranium in Fall River area, Clear Creek County, Colorado /

"October 15, 1951, [Site Issuance Date]."

Former Ocoya Landfill Site : Livingston County, Illinois.

Illinois EPA's initial evaluation of this site revealed problems such as erosion, exposed waste, low areas at the surface that allowed water to pond, and leachate seeps water that becomes contaminated after contact with landfill waste).

Little Butte/Bear Creek Subbasins Water Feasibility Act : report (to accompany S. 251).

"October 27, 2005."

Using Terrestrial LiDAR to Monitor Erosion within the Gold Basin Landslide Complex, Verlot, WA

In 2014 the United States Forest Service closed the Gold Basin Campground of western Washington in an effort to protect the public from unstable hillslopes directly adjacent to the campground. The Gold Basin Landslide Complex (GBLC) is actively eroding via block fall, dry ravel, and debris flows, which contribute sediment into the South Fork of the Stillaguamish River. This sediment diminishes the salmonid population within the South Fork of the Stillaguamish River by reducing habitable spawning grounds, which is a big concern to the Stillaguamish Tribe of Indians. In this investigation, I quantified patterns of degradation and total volume of sediment erosion from the middle lobe of the GBLC over the period of July 2015 through January 2016 using terrestrial (ground-based) LiDAR (TLS). I characterized site specific stratigraphy and geomorphic processes, and laid the groundwork for future, long-term monitoring of this site. Results of this investigation determined that ~ 4,800m3 of sediment was eroded from the middle lobe of the GBLC during the 6 month study period (July 2015 – January 2016). This erosion likely occurred from debris flows, raveling of poorly sorted sand and gravel deposits and block failures of high plasticity silts and clays, and/or other mass wasting mechanisms. The generalized stratigraphic sequence in the GBLC consists of alternating massive beds of sand and gravel with silts and clays. The low permeability of these silts and clays provide a perfect venue for groundwater to percolate, as I observed during field investigations, which likely contributes to the active instability of the hillslopes. Continued monitoring and mapping of this complex will lead to viable information that could help both the United States Forest Service and the Stillaguamish Tribe.

SITE 1121 - FIELD NOTES Revealing Deep Form as a Basis for Urban Landscape Design

Thesis (Master's)--University of Washington, 2016-06

Assessing the influence and distribution of shrimp pond effluent in a tidal mangrove creek in north-east Australia

Effluent from a land based shrimp farm was detected in a receiving creek as changes in physical, chemical and biological parameters. The extent and severity of these changes depended on farm operations. This assessment was conducted at three different stages of shrimp-pond maturity, including (1) when the ponds were empty, (2) full and (3) being harvested. Methods for assessing farm effluent in receiving waters included physical/chemical analyses of the water column, phytoplankton bioassays and nitrogen isotope signatures of marine flora. Comparisons were made with an adjacent creek that served as the farms intake creek and did not directly receive effluent. Physical/chemical parameters identified distinct changes in the receiving creek with respect to farm operations. Elevated water column NH4+ (18.5+/-8.0 muM) and chlorophyll a concentrations (5.5+/-1.9 mug/l) were measured when the farm was in operation, in contrast to when the farm was inactive (1.3+/-0.3 muM and 1.2+/-0.6 mug/l, respectively). At all times, physically chemical parameters at the mouth of the effluent creek, were equivalent to control values, indicating effluent was contained within the effluent-receiving creek. However, elevated delta(15)N signatures of mangroves (up to similar to8parts per thousand) and macroalgae (up to similar to5parts per thousand) indicated a broader influence of shrimp farm effluent, extending to the lower regions of the farms intake creek. Bioassays at upstream sites close to the location of farm effluent discharge indicated that phytoplankton at these sites did not respond to further nutrient additions, however downstream sites showed large growth responses. This suggested that further nutrient loading from the shrimp farm, resulting in greater nutrient dispersal, will increase the extent of phytoplankton blooms downstream from the site of effluent discharge. When shrimp ponds were empty water quality in the effluent and intake creeks was comparable. This indicated that observed elevated nutrient and phytoplankton concentrations were directly attributable to farm operations. (C) 2003 Elsevier Ltd. All rights reserved.