Functional and intracellular localization properties of U6 promoter-expressed siRNAs, shRNAs, and chimeric VA1 shRNAs in mammalian cells

RNA polymerase III (Pol III) expression systems for short hairpin RNAs (U6 shRNAs or chimeric VA1 shRNAs) or individually expressed sense/antisense small interfering RNA (siRNA) strands have been used to trigger RNA interference (RNAi) in mammalian cells. Here we show that individually expressed siRNA expression constructs produce 21-nucleotide siRNAs that strongly accumulate as duplex siRNAs in the nucleus of human cells, exerting sequence-specific silencing activity similar to cytoplasmic siRNAs derived from U6 or VA1-expressed hairpin precursors. In contrast, 29-mer siRNAs separately expressed as sense/antisense strands fail to elicit RNAi activity, despite accumulation of these RNAs in the nucleus. Our findings delineate different intracellular accumulation patterns for the three expression strategies and suggest the possibility of a nuclear RNAi pathway that requires 21-mer duplexes.     

Interaction of the chloroplast ATP synthetase with the photoreactive nucleotide 3′-O-(4-benzoyl)benzoyl adenosine 5''-diphosphate

The photoreactive nucleotide 3'-O-(4-benzoyl)benzoyl ADP (BzADP) is not a substrate for photophosphorylation but is a strong competitive inhibitor (K_i 2-25μM) with respect to ADP and ATP in photophosphorylation or ATP hydrolysis and P_i-ATP exchange reactions, respectively. The analog binds tightly to the membrane-bound CF₁, competes with the right binding of ADP, and prevents the inactivation of the enzyme by tight binding of ADP. Upon irradiation with long wavelength ultraviolet light, the tightly bound BzADP becomes covalently attached to both the - and β-subunits of the enzyme.     

Exploratory deep-sea fishing in the Falkland Islands, south-western Atlantic

Records of demersal deep-sea fish assemblages in waters around the Falkland Islands (Patagonian shelf area) are rare. Twenty deep-water stations to the east and south of the Falkland Islands were sampled by commercial bottom trawl deployed in upper, middle and lower benthopelagic zones (depth range of approximately 500-1000 m). Forty-one species (22 families) of teleost fish were recorded, 10 species (two families) of elasmobranch and one species of agnathan. Different assemblages of fish were found to characterize each depth zone (e.g. Moridae in deeper waters, Bothidae and Rajidae in shallower waters), with diversity being greatest in the mid-zone and biomass greatest in the upper and lower zones. Some species occurred in all zones but showed depth-related abundance. Four species, namely the grenadiers Macrourus carinutus and Coelorhynchus fasciatus , the southern blue whiting Micromesistius australis , and the Patagonian toothfish Dissostichus eleginoides , accounted for 85% by weight of all fish caught. Quantitative sampling of selected species revealed depth-related variations in their population structure. Length-frequency analyses are presented for M. carinatus and D. eleginoides and show a tendency for larger individuals to inhabit deeper water. Discard rates from the commercial catch were sometimes high, particularly for the smaller species, raising concerns about the impact of a fishery on by-catch species. The potential for deep-sea fisheries in Falkland waters is discussed and further studies are suggested in the light of developing oil, gas and fishing industries. The presence of some invertebrate taxa is recorded.     

Experimental induction and termination of non-reciprocal reproductive incompatibilities in a parahaploid mite

Two experiments were conducted to investigate the relationship between rearing temperatures, the presence or absence of Wolbachia endosymbionts, and non-reciprocal reproductive incompatibility in inbred lines of the parahaploid phytoseiid mite Metaseiulus occidentalis (Nesbitt) (Acari: Phytoseiidae).Heat-treated females crossed with infected males reared at room temperature produced few eggs and high proportions of shriveled eggs. No female progeny were produced. The reciprocal cross was normal. A second experiment showed that the incompatible cross from the first experiment could be made compatible if the infected line was heat-treated and those males crossed with the original heat-treated females. Furthermore, a new incompatibility was induced in a formerly compatible cross when the newly heat-treated females were crossed with males from their base colony. Heat-treatment was correlated with the loss of Wolbachia in both experiments. Wolbachia may thus affect non-reciprocal reproductive incompatibility in M. occidentalis, and may produce a unique incompatibility phenotype in this parahaploid species, including both reduced numbers of male progeny and a lack of female progeny.     

Phylogeography of the ground squirrel subgenus Xerospermophilus and assembly of the Mojave Desert biota

Aim The Mohave ground squirrel (Xerospermophilus mohavensis) is one of a few endemic species of the Mojave Desert of south‐western North America. We describe phylogeographic patterns within this species and its sister taxon (Xerospermophilus tereticaudus) and test hypotheses concerning their biogeographical history using genetic signatures of stable versus expanding populations. We compare these patterns with those of other Mojave species to evaluate the role of vicariance in producing phylogeographic structure during the assembly of the Mojave Desert biota. Location The Mojave Desert and adjacent desert regions of south‐western North America. Methods Complete cytochrome b gene sequences of X. mohavensis (46 individuals representing 11 localities) and X. tereticaudus (38 individuals representing 14 localities) were analysed using Bayesian methods to infer phylogenetic relationships. Genetic signals of stable or expanding populations were examined based on the distribution of recent mutations and pairwise differences, as well as with a coalescent‐based approach. Results The two species are reciprocally monophyletic and may have diverged in response to the late Pliocene–early Pleistocene uplift of the Transverse Ranges and Mojave block. Little phylogeographic structure is evident within X. mohavensis, but there is a signature of northern expansion from a presumably full‐pluvial refugium in the Mojave River basin. Four geographic subgroups are evident within X. tereticaudus, and there is a signature of northern expansion from a presumably full‐pluvial refugium in the Sonoran coastal plains. Roughly congruent phylogeographic patterns are found within five arid‐adapted taxa, indicating a strong element of vicariance during the assembly of the generally transitional Mojave Desert biota. Main conclusions We present a preliminary model for the historical assembly of the Mojave Desert biota that indicates a strong vicariant element producing autochthonous lineages (including X. mohavensis) that diverged during the major geological and climatic events of the last 5 Myr. Phylogeographic partitioning within the Mojave Desert underscores the necessity of immediate conservation measures for this unique and fragile arid ecosystem that is locked between two large metropolitan population centres and is the target of continued adverse environmental impact.     

Genome-wide genetic variation coupled with demographic and ecological niche modeling of the dusky-footed woodrat (Neotoma fuscipes) reveal patterns of deep divergence and widespread Holocene expansion across northern California

Understanding how species have responded to past climate change may help refine projections of how species and biotic communities will respond to future change. Here, we integrate estimates of genome-wide genetic variation with demographic and niche modeling to investigate the historical biogeography of an important ecological engineer: the dusky-footed woodrat, Neotoma fuscipes. We use RADseq to generate a genome-wide dataset for 71 individuals from across the geographic distribution of the species in California. We estimate population structure using several model-based methods and infer the demographic history of regional populations using a site frequency spectrum-based approach. Additionally, we use ecological niche modeling to infer current and past (Last Glacial Maximum) environmental suitability across the species’ distribution. Finally, we estimate the directionality and possible spatial origins of regional population expansions. Our analyses indicate this species is subdivided into three regionally distinct populations, with the deepest divergence occurring ~1.7 million years ago across the modern-day San Francisco-Bay Delta region; a common biogeographic barrier for the flora and fauna of California. Our models of environmental suitability through time coincide with our estimates of population expansion, with relative long-term stability in the southern portion of the range, and more recent expansion into the northern end of the range. Our study illustrates how the integration of genome-wide data with spatial and demographic modeling can reveal the timing and spatial extent of historic events that determine patterns of biotic diversity and may help predict biotic response to future change.Subject terms: Evolutionary genetics, Molecular evolution