An essential protein-binding domain of nuclear RNase P RNA

Eukaryotic RNase P and RNase MRP are endoribonucleases composed of RNA and protein subunits. The RNA subunits of each enzyme share substantial secondary structural features, and most of the protein subunits are shared between the two. One of the conserved RNA subdomains, designated P3, has previously been shown to be required for nucleolar localization. Phylogenetic sequence analysis suggests that the P3 domain interacts with one of the proteins common to RNase P and RNase MRP, a conclusion strengthened by an earlier observation that the essential domain can be interchanged between the two enzymes. To examine possible functions of the P3 domain, four conserved nucleotides in the P3 domain of Saccharomyces cerevisiae RNase P RNA (RPR1) were randomized to create a library of all possible sequence combinations at those positions. Selection of functional genes in vivo identified permissible variations, and viable clones that caused yeast to exhibit conditional growth phenotypes were tested for defects in RNase P RNA and tRNA biosynthesis. Under nonpermissive conditions, the mutants had reduced maturation of the RPR1 RNA precursor, an expected phenotype in cases where RNase P holoenzyme assembly is defective. This loss of RPR1 RNA maturation coincided, as expected, with a loss of pre-tRNA maturation characteristic of RNase P defects. To test whether mutations at the conserved positions inhibited interactions with a particular protein, specific binding of the individual protein subunits to the RNA subunit was tested in yeast using the three-hybrid system. Pop1p, the largest subunit shared by RNases P and MRP, bound specifically to RPR1 RNA and the isolated P3 domain, and this binding was eliminated by mutations at the conserved P3 residues. These results indicate that Pop1p interacts with the P3 domain common to RNases P and MRP, and that this interaction is critical in the maturation of RNase P holoenzyme.     

Biomass and distribution of fine and coarse roots from blue oak (Quercus douglasii) trees in the northern Sierra Nevada foothills of California

This research adds to the limited data on coarse and fine root biomass for blue oak (Quercus douglasii Hook and Arn.), a California deciduous oak species found extensively throughout the interior foothills surrounding the Central Valley. Root systems of six blue oak trees were analyzed using three methods — backhoe excavation, quantitative pits, and soil cores. Coarse root biomass ranged from 7 to 177 kg per tree. Rooting depth for the main root system ranged from 0.5 to 1.5 m, with an average of 70% of excavated root biomass located above 0.5 m. Of the total biomass in excavated central root systems, primary roots (including burls) accounted for 56% and large lateral roots (> 20 mm diameter) accounted for 36%. Data from cores indicated that most biomass outside of the root crown was located in fine roots and that fine root biomass decreased with depth. At surface depths (0–20 cm), small-fine (< 0.5 mm diameter) roots accounted for 71%, large-fine (0.5–2.0 mm) for 25%, and coarse (> 2 mm) for 4% of total root biomass collected with cores. Mean fine root biomass density in the top 50 cm was 0.43 kg m⁻³. Fine root biomass did not change with increasing distance from the trees (up to approximately 5 m). Thus, fine roots were not concentrated under the tree canopies. Our results emphasize the importance of the smallest size class of roots (<0.5 mm), which had both higher N concentration and, in the area outside the central root system, greater biomass than large fine (0.5–2.0 mm) or coarse (> 2.0 mm) roots. This revised version was published online in June 2006 with corrections to the Cover Date.     

Water‐borne and plasma corticosterone are not correlated in spotted salamanders

Water‐borne hormone measurement is a noninvasive method suitable for amphibians of all sizes that are otherwise difficult to sample. For this method, containment‐water is assayed for hormones released by the animal. Originally developed in fish, the method has expanded to amphibians, but requires additional species‐specific validations. We wanted to determine physiological relevance of water‐borne corticosterone in spotted salamanders (Ambystoma maculatum) by comparing concentrations to those taken using established corticosterone sampling methods, such as plasma. Using a mixture of field and laboratory studies, we compared water‐borne corticosterone levels to other traditional methods of sampling corticosterone for spotted salamander larvae, metamorphs, and adults. Despite multiple attempts, and detecting differences between age groups, we found no correlations between water‐borne and plasma corticosterone levels in any age group. Water‐borne sampling measures a rate of release; whereas plasma is the concentration circulating in the blood. The unique units of measurement may inherently prevent correlations between the two. These two methods may also require different interpretations of the data and the physiological meaning. We also note caveats with the method, including how to account for differences in body size and life history stages. Collectively, our results illustrate the importance of careful validation of water‐borne hormone levels in each species in order to understand its physiological significance.     

Identification and Quantification of Proteoforms by Mass Spectrometry

A proteoform is a defined form of a protein derived from a given gene with a specific amino acid sequence and localized post‐translational modifications. In top‐down proteomic analyses, proteoforms are identified and quantified through mass spectrometric analysis of intact proteins. Recent technological developments have enabled comprehensive proteoform analyses in complex samples, and an increasing number of laboratories are adopting top‐down proteomic workflows. In this review, some recent advances are outlined and current challenges and future directions for the field are discussed.     

Monitoring populations of Western Sandpipers and Pacific Dunlins during northward migration on the Fraser River Delta,British Columbia, 1991–2013

The Fraser River Delta in British Columbia, Canada, is a globally significant stopover site for shorebirds, but the population status and trends of many species that use the site remain uncertain. We describe an ongoing program to monitor population trends of the two most abundant species, Western Sandpipers (Calidris mauri) and Dunlins (Calidris alpina), during northward migration. Counts of these species were conducted at a mudflat where large flocks assembled at mid‐tide from 15 April to 15 May, 1991–2013, and we estimated species‐specific counts as the product of daily total flock counts and species proportions obtained during supplementary sampling. The median peak count of both species combined was 177,000 birds, and occurred between 24 April and 3 May. Ratios (proportions) of the two species followed a predictable pattern during the migration period, with a low proportion of Western Sandpipers (3%–20%) in flocks before 20 April, followed by a rapid increase to 80%–100% between 25 April and 10 May and a variable decrease to 30%–80% by 15 May. Mean counts of Western Sandpipers showed no significant trend over the study period. Mean counts of Dunlins showed a non‐linear trend, decreasing until 2001 and then increasing to 2013. Bias and random error in field counts were quantified by comparing field counts to counts made from photographs taken during surveys, and analysis revealed that field counts had a downward, but predictable, bias, accounting for >90% of birds present, with a stochastic error rate of 28.0%. Uncertainty in total population estimates was high after accounting for the effect of length of stay and sampling error. Population estimates suggested that 600,000 Western Sandpipers and 200,000 Dunlins typically passed through the site during northward migration. Our estimates indicate the usefulness of daily counts at major stopover sites during northward migration as an effective tool for monitoring shorebird populations, and underscore the need for conserving such sites.