Oligonucleotide Primers for PCR Amplification of Coelomate Introns

Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.     

Effect of Repeat Dosing of Engineered Oncolytic Herpes Simplex Virus on Preclinical Models of Rhabdomyosarcoma

Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, is the most common sarcoma of childhood. Despite multidrug chemotherapy regimens, surgical intervention, and radiation treatment, outcomes remain poor, especially in advanced disease, and novel therapies are needed for the treatment of these aggressive malignancies. Genetically engineered oncolytic viruses, such as herpes simplex virus-1 (HSV), are currently being explored as treatments for pediatric tumors. M002, an oncolytic HSV, has both copies of the γ₁34.5 gene deleted, enabling replication in tumor cells but thwarting infection of normal, postmitotic cells. We hypothesized that M002 would infect human RMS tumor cells and lead to decreased tumor cell survival in vitro and impede tumor growth in vivo. In the current study, we demonstrated that M002 could infect, replicate in, and decrease cell survival in both embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) cells. Additionally, M002 reduced xenograft tumor growth and increased animal survival in both ARMS and ERMS. Most importantly, we showed for the first time that repeated dosing of oncolytic virus coupled with low-dose radiation provided improved tumor response in RMS. These findings provide support for the clinical investigation of oncolytic HSV in pediatric RMS.     

Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

Unusual seasonal patterns and inferred processes of nitrogen retention in forested headwaters of the Upper Susquehanna River

Atmospheric deposition contributes a large fraction of the annual nitrogen (N) input to the basin of the Susquehanna River, a river that provides two-thirds of the annual N load to the Chesapeake Bay. Yet, there are few measurements of the retention of atmospheric N in the Upper Susquehanna’s forested headwaters. We characterized the amount, form (nitrate, ammonium, and dissolved organic nitrogen), isotopic composition (δ¹⁵N- and δ¹⁸O-nitrate), and seasonality of stream N over 2 years for 7–13 catchments. We expected high rates of N retention and seasonal nitrate patterns typical of other seasonally snow-covered catchments: dormant season maxima and growing season minima. Coarse estimates of N export indicated high rates of inorganic N retention (>95%), yet streams had unexpected seasonal nitrate patterns, with summer peaks (14–96 μmol L⁻¹), October crashes (<1 μmol L⁻¹), and modest rebounds during the dormant season (<1–20 μmol L⁻¹). Stream δ¹⁸O-nitrate values indicated microbial nitrification as the primary source of stream nitrate, although snowmelt or other atmospheric source contributed up to 47% of stream nitrate in some March samples. The autumn nitrate crash coincided with leaffall, likely due to in-stream heterotrophic uptake of N. Hypothesized sources of the summer nitrate peaks include: delayed release of nitrate previously flushed to groundwater, weathering of geologic N, and summer increases in net nitrate production. Measurements of shale δ¹⁵N and soil-, well-, and streamwater nitrate within one catchment point toward a summer increase in soil net nitrification as the driver of this pattern. Rather than seasonal plant demand, processes governing the seasonal production, retention, and transport of nitrate in soils may drive nitrate seasonality in this and many other systems.     

Kinetics of 3-O-methyl-D-glucose transport in isolated rat hepatocytes.

3-O-Methyl-D-glucose transport across the plasma membrane of isolated rat hepatocytes was followed for net entry of the sugar into sugar-free cells (zero trans entry), net exit of sugar into sugar-free medium (zero trans exit) and for unidirectional entry and exit fluxes when cells had been equilibrated with sugar in the extracellular medium (equilibrium exchange entry and exit). These measurements were performed at 20 degrees C and pH 7.4 by the use of simple manual methods. Initial rates of transport showed a Michaelis--Menten dependency on the sugar concentration at the cis side of the membrane over the range of concentrations tested (100 microM to 100 mM). Transport was found to be symmetrical with no evidence of substrate stimulation of transport from the trans side of the membrane. Parameters (mean values +/- S.E.M.) of transport were estimated as Vmax. 86.2 +/- 9.7 mmol/litre of cell water per min and Km 18.1 +/- 5.9 mM for exchange entry, Vmax. 78.8 +/- 5.3 mmol/litre of cell water per min and Km 17.6 +/- 3.5 mM for exchange exit, Vmax. 84.1 +/- 8.4 mmol/litre of cell water per min and Km 16.8 +/- 4.6 mM for zero trans exit.     

Variation analysis and gene annotation of eight MHC haplotypes: The MHC Haplotype Project

The human major histocompatibility complex (MHC) is contained within about 4 Mb on the short arm of chromosome 6 and is recognised as the most variable region in the human genome. The primary aim of the MHC Haplotype Project was to provide a comprehensively annotated reference sequence of a single, human leukocyte antigen-homozygous MHC haplotype and to use it as a basis against which variations could be assessed from seven other similarly homozygous cell lines, representative of the most common MHC haplotypes in the European population. Comparison of the haplotype sequences, including four haplotypes not previously analysed, resulted in the identification of >44,000 variations, both substitutions and indels (insertions and deletions), which have been submitted to the dbSNP database. The gene annotation uncovered haplotype-specific differences and confirmed the presence of more than 300 loci, including over 160 protein-coding genes. Combined analysis of the variation and annotation datasets revealed 122 gene loci with coding substitutions of which 97 were non-synonymous. The haplotype (A3-B7-DR15; PGF cell line) designated as the new MHC reference sequence, has been incorporated into the human genome assembly (NCBI35 and subsequent builds), and constitutes the largest single-haplotype sequence of the human genome to date. The extensive variation and annotation data derived from the analysis of seven further haplotypes have been made publicly available and provide a framework and resource for future association studies of all MHC-associated diseases and transplant medicine. Horton and Gibson contributed equally to this work.     

Network isolation and local diversity in neutral metacommunities

Biologists seek an understanding of the biological and environmental factors determining local community diversity. Recent advances in metacommunity ecology, and neutral theory in particular, highlight the importance of dispersal processes interacting with the spatial structure of a landscape for generating spatial patterns and maintaining biodiversity. The relative spatial isolation of a community is traditionally thought to have a large influence on local diversity. However, isolation remains an elusive concept to quantify, particularly in metacommunities with complex spatial structure. We represent the metacommunity as a network of local communities, and use network centrality measures to quantify the isolation of a local community. Using spatially explicit neutral theory, we examine how node position predicts variation in alpha diversity across a metacommunity. We find that diversity increases with node centrality in the network, but only when centrality is measured on a given scale in the network that widens with increasing dispersal rates and narrows with increasing evolutionary rates. More generally, complex biodiversity patterns form only when the underlying geography has structure on this critical scale. This provides a framework for understanding the influence of spatial geographic structure on global biodiversity patterns.