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Capturing the 5′ end of transcripts from an input of only 1,000 cells and linking the transcription start sites to downstream sequences allows the analysis of complex transcriptome architecture.
A set of computational and imaging approaches, called MARS-ALT, permits three-dimensional tracking of plant tissue development, including cell lineaging, at cellular resolution. It is applied to the study of floral development in Arabidopsis.
A method to analyze the sequence of C-terminal peptides using a combination of a specific enrichment approach and mass spectrometry is described, allowing the study of C-terminal proteolytic processing on a global scale. Also in this issue, Van Damme et al. describe a related method for simultaneous C- and N-terminal peptide analysis.
A method to simultaneously analyze C- and N-terminal peptides using a combination of strong cation exchange, diagonal chromatography and mass spectrometry is described, allowing the screening of protease substrates on a global scale. Also in this issue, Schilling et al. describe a related method for analyzing the sequence of C-terminal peptides.
This technique allows functional imaging of neurons in head-fixed Drosophila while the fly walks on an air-supported ball. Using a genetically encoded calcium sensor, the activity of motion-sensitive neurons in the fly optic lobe was recorded while the flies were presented with visual stimuli. Activity in these cells correlated with robust optomotor behavior in the walking flies.
The misfolded form of the prion protein, PrPSc, can be quantified in a variety of tissues and fluids using a quantitative version of the popular protein misfolding cyclic amplification (PMCA) assay.
Expression of the transporter SID-1 in Caenorhabditis elegans neurons renders the cells sensitive to systemic RNAi and permits previously unidentified neuronal phenotypes to be uncovered. This expression also reduces RNAi in nonneuronal cell types, allowing examination of neuronal functions of lethal genes.
By micropatterning cells in polarized shapes the global distribution of endomembranes can be compared and subtle but statistically significant changes can be detected from only tens of cells.
This software integrates data from multiple samples and single-nucleotide polymorphism and comparative genome hybridization array platforms to detect and genotype copy-number variants (CNVs). It has high accuracy in the detection of short deletions and amplifications and will be valuable for genome-wide CNV analyses in population studies.
Paired-end reads consisting of 5′ transcription start sites and 3′ downstream sequences from transcripts in Drosophila melanogaster reveal distinct initiation patterns at different fly promoters and show that 5′ caps originating in coding regions are added posttranscriptionally.
Cultured rat spermatogonia, genetically modified via a transposon-based gene trap, are inserted into the testes of sterile rats and give rise to mutant progeny with many different genotypes.
A photoconvertible reporter of the ubiquitin-proteasome system permits detection of its activity independent of protein synthesis and is applied to study cell type– and age-specific protein degradation in living Caenorhabditis elegans.
Polymerase kinetics observed during single-molecule, real-time sequencing depend on the methylation status of the DNA template. Measurement of kinetic parameters such as interpulse duration and pulse width allows the identification of methylated adenosine in Escherichia coli and the distinction between 5-methylcytosine and 5-hydroxymethylcytosine in synthetic templates.
Combining reverse transfection of protein tyrosine kinase substrates on cell arrays with fluorescence resonance energy transfer (FRET) measurements by fluorescence lifetime imaging microscopy (FLIM) allows quantitative assessment of phosphorylation patterns and identification of feedback loops at single-cell resolution.
The activity of zinc-finger nucleases in mammalian cells is enhanced by transient incubation of the cells at low temperatures. Incorporation of this simple step should improve the efficiency of these tools for genome manipulation.
This computational process evaluates gene models in prokaryotic genomes, independently of the gene finder used, and reports anomalies that can be used to improve the quality of gene models through manual curation.
Targeted deletion of genes within 25 kb of Mos1 transposons in the C. elegans genome is demonstrated. This should enable single-gene deletions of more than 8,000 genes for which deletions are currently not available in this organism.
Paired-end sequencing of human genomic DNA reveals at least 2.8 Mb of new sequence at 720 distinct loci. Complete sequencing of 1.67 Mb at 192 loci reveals extensive copy-number variation and provides a resource for genotyping these 'missing' sequences.
Limitations in scanning speed have made it difficult for two-photon imaging to provide accurate temporal information on neuronal signaling. Refinements to random-access scanning using acousto-optic deflectors and an automated algorithm for reconstructing complex spike trains allowed in vivo high-speed optical recording of spiking activity in neuronal populations in the mouse neocortex.
