The systematic comparison of genomic sequences from different organisms represents a central focus of contemporary genome analysis. Comparative analyses of vertebrate sequences can identify coding and conserved non-coding regions, including regulatory elements, and provide insight into the forces that have rendered modern-day genomes. As a complement to whole-genome sequencing efforts, we are sequencing and comparing targeted genomic regions in multiple, evolutionarily diverse vertebrates. Here we report the generation and analysis of over 12 megabases (Mb) of sequence from 12 species, all derived from the genomic region orthologous to a segment of about 1.8 Mb on human chromosome 7 containing ten genes, including the gene mutated in cystic fibrosis. These sequences show conservation reflecting both functional constraints and the neutral mutational events that shaped this genomic region. In particular, we identify substantial numbers of conserved non-coding segments beyond those previously identified experimentally, most of which are not detectable by pair-wise sequence comparisons alone. Analysis of transposable element insertions highlights the variation in genome dynamics among these species and confirms the placement of rodents as a sister group to the primates.

Research into the genetic basis of the ecological sophistication of plants is hampered by the availability of transformable systems with a wealth of well-described ecological interactions. We present an Agrobacterium-mediated transformation system for the model ecological expression system, Nicotiana attenuata, a native tobacco that occupies the post-fire niche in the Great Basin Desert of North America. We describe a transformation vector and a transformation procedure that differs from the standard cultivated tobacco transformation protocols in its use of selectable markers, explants, media and cultivation conditions. We illustrate its utility in the transformations with genes coding for key enzymes in the oxylipin cascade (lipoxygenase, allene oxide synthase, hydroperoxide lyase) in antisense orientations and present high-throughput screens useful for the detection of altered phenotypes for the oxylipin cascade (green leaf volatiles and jasmonic acid after wounding).

Transfer RNA (tRNA)‐derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell‐state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation‐dependent enrichment of 5′‐tsRNAs. We report the identification of a set of 5′‐tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5′‐tsRNAs revealed a switch in their association with “effector” RNPs and “target” mRNAs in different cell states. We demonstrate that specific 5′‐tsRNAs can preferentially interact with the RNA‐binding protein, Igf2bp1, in the RA‐induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency‐promoting factor, c‐Myc, thus providing a mechanistic basis for how 5′‐tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5′‐tsRNAs in defining distinct cell states.

Plants respond to environmental stresses through a series of complicated phenotypic responses, which can be understood only with field studies because other organisms must be recruited for their function. If ecologists are to fully participate in the genomics revolution and if molecular biologists are to understand adaptive phenotypic responses, native plant ecological expression systems that offer both molecular tools and interesting natural histories are needed. Here, we present Solanum nigrum L., a Solanaceous relative of potato and tomato for which many genomic tools are being developed, as a model plant ecological expression system. To facilitate manipulative ecological studies with S. nigrum , we describe: (i) an Agrobacterium -based transformation system and illustrate its utility with an example of the antisense expression of RuBPCase , as verified by Southern gel blot analysis and realtime quantitative PCR; (ii) a 789-oligonucleotide microarray and illustrate its utility with hybridizations of herbivore-elicited plants, and verify responses with RNA gel blot analysis and real-time quantitative PCR; (iii) analyses of secondary metabolites that function as direct (proteinase inhibitor activity) and indirect (herbivore-induced volatile organic compounds) defences; and (iv) growth and fitness-estimates for plants grown under field conditions. Using these tools, we demonstrate that attack from flea beetles elicits: (i) a large transcriptional change consistent with elicitation of both jasmonate and salicylate signalling; and (ii) increases in proteinase inhibitor transcripts and activity, and volatile organic compound release. Both flea beetle attack and jasmonate elicitation increased proteinase inhibitors and jasmonate elicitation decreased fitness in field-grown plants. Hence, proteinase inhibitors and jasmonate-signalling are targets for manipulative studies.

Analysis of single-cell RNA-seq data is challenging due to technical variability, high noise levels and massive sample sizes. Here, we describe a normalization technique that substantially reduces technical variability and improves the quality of downstream analyses. We also introduce a nonparametric method for detecting differentially expressed genes that scales to > 1, 000 cells and is both more accurate and ∼ 10 times faster than existing parametric approaches.