Use of Vertical Slab Isoelectric Focusing and Immunoblotting to Evaluate Steady-State Phosphorylation of eIF2α in Cultured Cells

The combination of vertical, one-dimensional isoelectric focusing and immunoblotting works very well for the evaluation of the phosphorylation state of the α-subunit of eIF2 using reticulocyte lysate or purified eIF2. However, the method is more difficult to apply to the analysis of eIF2α phosphorylation in cultured cells. In part this reflects the fact that the protein content of cultured cell extracts is rarely as high as that found in extracts produced from reticulocytes, and in part this reflects the fact that some component(s) of cell extracts interferes with the entry of eIF2α into the isoelectric focusing gel. To overcome these difficulties, we have modified the earlier method to include immunoprecipitation of eIF2 from cell extracts prior to isoelectric focusing, as well as a low sodium dodecyl sulfate concentration in the isoelectric focusing sample buffer. Since the PKR activation state and therefore the eIF2α phosphorylation state change with cell density and nutritional status, we routinely set up consistent feeding schedules and recommend the collection of data over a range of cell densities.     

Confident phylogenetic identification of uncultured prokaryotes through long read amplicon sequencing of the 16S-ITS-23S rRNA operon

Amplicon sequencing of the 16S rRNA gene is the predominant method to quantify microbial compositions and to discover novel lineages. However, traditional short amplicons often do not contain enough information to confidently resolve their phylogeny. Here we present a cost-effective protocol that amplifies a large part of the rRNA operon and sequences the amplicons with PacBio technology. We tested our method on a mock community and developed a read-curation pipeline that reduces the overall read error rate to 0.18%. Applying our method on four environmental samples, we captured near full-length rRNA operon amplicons from a large diversity of prokaryotes. The method operated at moderately high-throughput (22286–37,850 raw ccs reads) and generated a large amount of putative novel archaeal 23S rRNA gene sequences compared to the archaeal SILVA database. These long amplicons allowed for higher resolution during taxonomic classification by means of long (∼1000 bp) 16S rRNA gene fragments and for substantially more confident phylogenies by means of combined near full-length 16S and 23S rRNA gene sequences, compared to shorter traditional amplicons (250 bp of the 16S rRNA gene). We recommend our method to those who wish to cost-effectively and confidently estimate the phylogenetic diversity of prokaryotes in environmental samples at high throughput.     

Dredge and fill regulatory constraints in meeting the ecological goals of restoration projects

Regulators face the problem of ensuring that wetland functions that are lost due to permitting actions are restored via mitigation of those impacts. While there has been considerable study of wetland function, little of it has been directed toward the practical assessment of mitigation success. Development of quantitatively based rapid assessment methods would provide valuable assistance to the regulatory community. Regional and local goal setting, including a determination of whether strict in-kind restoration or restoration of habitat balance is preferable, is needed to ensure that the ecological health of the area is restored to the greatest extent possible.     

Bootstrap, Bayesian probability and maximum likelihood mapping: exploring new tools for comparative genome analyses

Background  

Horizontal gene transfer (HGT) played an important role in shaping microbial genomes. In addition to genes under sporadic selection, HGT also affects housekeeping genes and those involved in information processing, even ribosomal RNA encoding genes. Here we describe tools that provide an assessment and graphic illustration of the mosaic nature of microbial genomes.     

Some sampling considerations in the quantitation of monkey behavior under field and captive conditions

Observations of monkey behavior were made on free-ranging and corral-enclosedMacaca mulatta. Two sampling techniques were used: focal animal and group observations. Group observations measured frequencies of social behavior whereas focal observations used time samples which estimated durations of social and nonsocial activities as well as frequencies of social behavior. Analysis of concordance within and between sampling techniques revealed that short duration focal time samples provide a reliable estimate of total frequencies of social behaviors derived using group observations and furthermore, allows the recording of important nonsocial activities not easily recorded in terms of frequencies. Focal time sampling is advantageous for certain types of studies where the equal distribution of observation time between individuals is important, particularly in the field where visibility is often limited and individuals can only be observed continuously for short periods of time. This work was supported by grants from the H. F. Guggenheim Foundation, Dr.H. C. Robinson, Jr., and USPHS grants MH 24607 and MH 25642.     

Differentiation of subspecies and sexes of Beringian Dunlins using morphometric measures

Five subspecies of Dunlins (Calidris alpina) that breed in Beringia are potentially sympatric during the non‐breeding season. Studying their ecology during this period requires techniques to distinguish individuals by subspecies. Our objectives were to determine (1) if five morphometric measures (body mass, culmen, head, tarsus, and wing chord) differed between sexes and among subspecies (C. a. actites, arcticola, kistchinski, pacifica, and sakhalina), and (2) if these differences were sufficient to allow for correct classification of individuals using equations derived from discriminant function analyses. We conducted analyses using morphometric data from 10 Dunlin populations breeding in northern Russia and Alaska, USA. Univariate tests revealed significant differences between sexes in most morphometric traits of all subspecies, and discriminant function equations predicted the sex of individuals with an accuracy of 83–100% for each subspecies. We provide equations to determine sex and subspecies of individuals in mixed subspecies groups, including the (1) Western Alaska group of arcticola and pacifica (known to stage together in western Alaska) and (2) East Asia group of arcticola, actites, kistchinski, and sakhalina (known to winter together in East Asia). Equations that predict the sex of individuals in mixed groups had classification accuracies between 75% and 87%, yielding reliable classification equations. We also provide equations that predict the subspecies of individuals with an accuracy of 22–96% for different mixed subspecies groups. When the sex of individuals can be predetermined, the accuracy of these equations is increased substantially. Investigators are cautioned to consider limitations due to age and feather wear when using these equations during the non‐breeding season. These equations will allow determination of sexual and subspecies segregation in non‐breeding areas, allowing implementation of taxonomic‐specific conservation actions.     

Identification of ATP-Binding Regions in the RyR1 Ca2+ Release Channel

ATP is an important modulator of gating in type 1 ryanodine receptor (RyR1), also known as a Ca²⁺ release channel in skeletal muscle cells. The activating effect of ATP on this channel is achieved by directly binding to one or more sites on the RyR1 protein. However, the number and location of these sites have yet to be determined. To identify the ATP-binding regions within RyR1 we used 2N₃ATP-2′,3′-Biotin-LC-Hydrazone (BioATP-HDZ), a photo-reactive ATP analog to covalently label the channel. We found that BioATP-HDZ binds RyR1 specifically with an IC₅₀ = 0.6±0.2 mM, comparable with the reported EC50 for activation of RyR1 with ATP. Controlled proteolysis of labeled RyR1 followed by sequence analysis revealed three fragments with apparent molecular masses of 95, 45 and 70 kDa that were crosslinked by BioATP-HDZ and identified as RyR1 sequences. Our analysis identified four glycine-rich consensus motifs that can potentially constitute ATP-binding sites and are located within the N-terminal 95-kDa fragment. These putative nucleotide-binding sequences include amino acids 699–704, 701–706, 1081–1084 and 1195–1200, which are conserved among the three RyR isoforms. Located next to the N-terminal disease hotspot region in RyR1, these sequences may communicate the effects of ATP-binding to channel function by tuning conformational motions within the neighboring cytoplasmic regulatory domains. Two other labeled fragments lack ATP-binding consensus motifs and may form non-canonical ATP-binding sites. Based on domain topology in the 3D structure of RyR1 it is also conceivable that the identified ATP-binding regions, despite their wide separation in the primary sequence, may actually constitute the same non-contiguous ATP-binding pocket within the channel tetramer.