Showing papers in "Nature Methods in 2009"
TL;DR: An isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5′ exonuclease, a DNA polymerase and a DNA ligase is described.
Abstract: We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
8,117 citations
TL;DR: A method is described, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics and allows single-run analyses of organelles and an unprecedented depth of proteome coverage.
Abstract: A method, filter-aided sample preparation (FASP) combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics, allowing deeper proteomic coverage in a shorter analysis time, using small sample amounts. We describe a method, filter-aided sample preparation (FASP), which combines the advantages of in-gel and in-solution digestion for mass spectrometry–based proteomics. We completely solubilized the proteome in sodium dodecyl sulfate, which we then exchanged by urea on a standard filtration device. Peptides eluted after digestion on the filter were pure, allowing single-run analyses of organelles and an unprecedented depth of proteome coverage.
6,096 citations
TL;DR: A single-cell digital gene expression profiling assay with only a single mouse blastomere is described, which detected the expression of 75% more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads.
Abstract: Next-generation sequencing technology is a powerful tool for transcriptome analysis. However, under certain conditions, only a small amount of material is available, which requires more sensitive techniques that can preferably be used at the single-cell level. Here we describe a single-cell digital gene expression profiling assay. Using our mRNA-Seq assay with only a single mouse blastomere, we detected the expression of 75% (5,270) more genes than microarray techniques and identified 1,753 previously unknown splice junctions called by at least 5 reads. Moreover, 8-19% of the genes with multiple known transcript isoforms expressed at least two isoforms in the same blastomere or oocyte, which unambiguously demonstrated the complexity of the transcript variants at whole-genome scale in individual cells. Finally, for Dicer1(-/-) and Ago2(-/-) (Eif2c2(-/-)) oocytes, we found that 1,696 and 1,553 genes, respectively, were abnormally upregulated compared to wild-type controls, with 619 genes in common.
2,659 citations
TL;DR: A single-wavelength GCaMP2-based GECI (GCaMP3) is developed, with increased baseline fluorescence, increased dynamic range and higher affinity for calcium, and long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.
Abstract: Genetically encoded calcium indicators (GECIs) can be used to image activity in defined neuronal populations. However, current GECIs produce inferior signals compared to synthetic indicators and recording electrodes, precluding detection of low firing rates. We developed a single-wavelength GCaMP2-based GECI (GCaMP3), with increased baseline fluorescence (3-fold), increased dynamic range (3-fold) and higher affinity for calcium (1.3-fold). We detected GCaMP3 fluorescence changes triggered by single action potentials in pyramidal cell dendrites, with signal-to-noise ratio and photostability substantially better than those of GCaMP2, D3cpVenus and TN-XXL. In Caenorhabditis elegans chemosensory neurons and the Drosophila melanogaster antennal lobe, sensory stimulation-evoked fluorescence responses were significantly enhanced with GCaMP3 (4-6-fold). In somatosensory and motor cortical neurons in the intact mouse, GCaMP3 detected calcium transients with amplitudes linearly dependent on action potential number. Long-term imaging in the motor cortex of behaving mice revealed large fluorescence changes in imaged neurons over months.
1,862 citations
TL;DR: In this article, a method for probing a target sequence of messenger ribonucleic acid molecules (mRNA's) in a fixed, permeabilized cell, including at least 30 non- overlapping probe binding regions of 15-100 nucleotides, was proposed.
Abstract: A method for probing a target sequence of messenger ribonucleic acid molecules (mRNA's) in a fixed, permeabilized cell, said target sequence including at least 30 non- overlapping probe binding regions of 15-100 nucleotides, comprising immersing said cell in an excess of at least 30 nucleic acid hybridization probes, each singly labeled with the same fluorescent label and each containing a nucleic acid sequence that is complementary to a different probe binding region of said target sequence; washing said fixed cell to remove unbound probes; and detecting fluorescence from said probes.
1,480 citations
TL;DR: The algorithm BreakDancer predicts a wide variety of structural variants including insertion-deletions (indels), inversions and translocations and sensitively and accurately detected indels ranging from 10 base pairs to 1 megabase pair that are difficult to detect via a single conventional approach.
Abstract: This software package provides genome-wide detection of structural variants (insertions, deletions, inversions and inter- and intrachromosomal translocations) from 50-base-pair paired-end reads. The sizes of the detected variants vary from 10 base pairs to 1 megabase pair.
1,418 citations
TL;DR: The auxin-inducible degron (AID) system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function.
Abstract: Plants have evolved a unique system in which the plant hormone auxin directly induces rapid degradation of the AUX/IAA family of transcription repressors by a specific form of the SCF E3 ubiquitin ligase Other eukaryotes lack the auxin response but share the SCF degradation pathway, allowing us to transplant the auxin-inducible degron (AID) system into nonplant cells and use a small molecule to conditionally control protein stability The AID system allowed rapid and reversible degradation of target proteins in response to auxin and enabled us to generate efficient conditional mutants of essential proteins in yeast as well as cell lines derived from chicken, mouse, hamster, monkey and human cells, thus offering a powerful tool to control protein expression and study protein function
1,335 citations
TL;DR: A nonradioactive fluorescence-activated cell sorting–based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations.
Abstract: We developed a nonradioactive fluorescence-activated cell sorting-based assay, called surface sensing of translation (SUnSET), which allows the monitoring and quantification of global protein synthesis in individual mammalian cells and in heterogeneous cell populations. We demonstrate here, using mouse dendritic and T cells as a model, that SUnSET offers a technical alternative to classical radioactive labeling methods for the study of mRNA translation and cellular activation.
1,220 citations
TL;DR: An algorithm is presented, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise and infers the true sequences in a collection of amplicons.
Abstract: We present an algorithm, PyroNoise, that clusters the flowgrams of 454 pyrosequencing reads using a distance measure that models sequencing noise. This infers the true sequences in a collection of amplicons. We pyrosequenced a known mixture of microbial 16S rDNA sequences extracted from a lake and found that without noise reduction the number of operational taxonomic units is overestimated but using PyroNoise it can be accurately calculated.
1,028 citations
TL;DR: The principles of these methods together with their differences in implementation and operation are discussed, and potential developments are outlined.
Abstract: Until not very long ago, it was widely accepted that lens-based (far-field) optical microscopes cannot visualize details much finer than about half the wavelength of light. The advent of viable physical concepts for overcoming the limiting role of diffraction in the early 1990s set off a quest that has led to readily applicable and widely accessible fluorescence microscopes with nanoscale spatial resolution. Here I discuss the principles of these methods together with their differences in implementation and operation. Finally, I outline potential developments.
978 citations
TL;DR: The results indicate that high-throughput yeast two-hybrid interactions for human proteins are more precise than literature-curated interactions supported by a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome.
Abstract: Several attempts have been made to systematically map protein-protein interaction, or 'interactome', networks. However, it remains difficult to assess the quality and coverage of existing data sets. Here we describe a framework that uses an empirically-based approach to rigorously dissect quality parameters of currently available human interactome maps. Our results indicate that high-throughput yeast two-hybrid (HT-Y2H) interactions for human proteins are more precise than literature-curated interactions supported by a single publication, suggesting that HT-Y2H is suitable to map a significant portion of the human interactome. We estimate that the human interactome contains approximately 130,000 binary interactions, most of which remain to be mapped. Similar to estimates of DNA sequence data quality and genome size early in the Human Genome Project, estimates of protein interaction data quality and interactome size are crucial to establish the magnitude of the task of comprehensive human interactome mapping and to elucidate a path toward this goal.
TL;DR: Fitness for each gene of Streptococcus pneumoniae, a causative agent of pneumonia and meningitis is determined using a method based on the assembly of a saturated Mariner transposon insertion library.
Abstract: High-throughput sequencing of Mariner transposon insertion libraries is used for quantitative studies of fitness and of genetic interactions in Streptococcus pneumoniae. The approach should allow similar studies in several microorganismal species.
TL;DR: A camera-based method for automatically quantifying the individual and social behaviors of fruit flies, Drosophila melanogaster, interacting in a planar arena finds that behavioral differences between individuals were consistent over time and were sufficient to accurately predict gender and genotype.
Abstract: We present a camera-based method for automatically quantifying the individual and social behaviors of fruit flies, Drosophila melanogaster, interacting in a planar arena Our system includes machine-vision algorithms that accurately track many individuals without swapping identities and classification algorithms that detect behaviors The data may be represented as an ethogram that plots the time course of behaviors exhibited by each fly or as a vector that concisely captures the statistical properties of all behaviors displayed in a given period We found that behavioral differences between individuals were consistent over time and were sufficient to accurately predict gender and genotype In addition, we found that the relative positions of flies during social interactions vary according to gender, genotype and social environment We expect that our software, which permits high-throughput screening, will complement existing molecular methods available in Drosophila, facilitating new investigations into the genetic and cellular basis of behavior
TL;DR: A high-speed structured-illumination microscope capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points is demonstrated by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
Abstract: Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.
TL;DR: FASP method is described as “universal” for its ability to represent the proteome in an unbiased way, which was demonstrated by comparison to the transcriptome by demonstrating a disproportionate reduction in peptide ion current, peptide or protein identifications.
Abstract: To the Editor: Large sequence-based datasets are often scanned for conserved sequence patterns to extract useful biological information1. Sequence logos2 have been developed to visualize conserved patterns in oligonucleotide and protein sequences and rely on enabled us to combine the advantages of in-gel and in-solution digestion workflows. It has been commonly held that SDS, once introduced into the sample, will make subsequent mass spectrometric analysis impossible. Manza et al.3 explicitly state in their paper that they could not completely remove SDS and that its presence reduced the number of identified BSA peptides. Indeed, after multidimensional separation Manza et al.3 identified 75 soluble cytosolic and 142 nuclear proteins. In contrast, our FASP approach allowed us to identify more than 7,000 proteins, about one-third of which were membrane or membrane-associated proteins2. In FASP, SDS is dissociated from proteins using urea. This presumably sequesters them into small micelles, which can pass through the filter pores, thus separating protein and detergent. The method of Manza et al.3 does not use such a step and is therefore not effective at removing SDS or other detergents. FASP achieves essentially complete protein unfolding during the whole process of detergent removal, which allows use of largemolecular-weight cut-off filters without a loss of small proteins. In contrast, Manza et al.3 reported that it was necessary to limit filter size to the 3–5 kDa range. The ability of FASP to work with larger pore filters substantially reduces sample preparation time. Liebler and Ham1 also state that the method is not “universal” because it disproportionately loses protein at low sample loads. We did not specifically develop the FASP protocol for high-sensitivity work. However, we demonstrated identification of 1,700 proteins from HeLa cell material corresponding to only 1,250 cells (750 ng total protein)2. We have now tested FASP with tenfold lower amounts and did not observe a disproportionate reduction in peptide ion current, peptide or protein identifications (Supplementary Note). Current commercial spin filters are not optimized for FASP, and they are not optimal for working with very small protein amounts (<100 ng). Miniaturization of the filter units should reduce proteins losses proportionally. In any case, in describing FASP method as “universal,” we were specifically referring to its ability to represent the proteome in an unbiased way, which we demonstrated by comparison to the transcriptome.
TL;DR: An amplification-free method of library preparation is presented, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly.
Abstract: Amplification artifacts introduced during library preparation for the Illumina Genome Analyzer increase the likelihood that an appreciable proportion of these sequences will be duplicates and cause an uneven distribution of read coverage across the targeted sequencing regions. As a consequence, these unfavorable features result in difficulties in genome assembly and variation analysis from the short reads, particularly when the sequences are from genomes with base compositions at the extremes of high or low G+C content. Here we present an amplification-free method of library preparation, in which the cluster amplification step, rather than the PCR, enriches for fully ligated template strands, reducing the incidence of duplicate sequences, improving read mapping and single nucleotide polymorphism calling and aiding de novo assembly. We illustrate this by generating and analyzing DNA sequences from extremely (G+C)-poor (Plasmodium falciparum), (G+C)-neutral (Escherichia coli) and (G+C)-rich (Bordetella pertussis) genomes.
TL;DR: A digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing is developed.
Abstract: The orchestrated binding of transcriptional activators and repressors to specific DNA sequences in the context of chromatin defines the regulatory program of eukaryotic genomes. We developed a digital approach to assay regulatory protein occupancy on genomic DNA in vivo by dense mapping of individual DNase I cleavages from intact nuclei using massively parallel DNA sequencing. Analysis of >23 million cleavages across the Saccharomyces cerevisiae genome revealed thousands of protected regulatory protein footprints, enabling de novo derivation of factor binding motifs and the identification of hundreds of new binding sites for major regulators. We observed striking correspondence between single-nucleotide resolution DNase I cleavage patterns and protein-DNA interactions determined by crystallography. The data also yielded a detailed view of larger chromatin features including positioned nucleosomes flanking factor binding regions. Digital genomic footprinting should be a powerful approach to delineate the cis-regulatory framework of any organism with an available genome sequence.
TL;DR: P piggyBac transposon–based reprogramming may be used to generate therapeutically applicable iPSCs and could be identified by negative selection.
Abstract: Induced pluripotent stem cells (iPSCs) have been generated from somatic cells by transgenic expression of Oct4 (Pou5f1), Sox2, Klf4 and Myc. A major difficulty in the application of this technology for regenerative medicine, however, is the delivery of reprogramming factors. Whereas retroviral transduction increases the risk of tumorigenicity, transient expression methods have considerably lower reprogramming efficiencies. Here we describe an efficient piggyBac transposon-based approach to generate integration-free iPSCs. Transposons carrying 2A peptide-linked reprogramming factors induced reprogramming of mouse embryonic fibroblasts with equivalent efficiencies to retroviral transduction. We removed transposons from these primary iPSCs by re-expressing transposase. Transgene-free iPSCs could be identified by negative selection. piggyBac excised without a footprint, leaving the iPSC genome without any genetic alteration. iPSCs fulfilled all criteria of pluripotency, such as pluripotency gene expression, teratoma formation and contribution to chimeras. piggyBac transposon-based reprogramming may be used to generate therapeutically applicable iPSCs.
TL;DR: A chemical approach is described that dramatically improves the efficiency of iPSC generation from human fibroblasts, within seven days of treatment, which will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.
Abstract: A cocktail of three small molecules improves the efficiency of reprogramming human fibroblasts to induced pluripotent stem cells and allows survival of the cells after trypsinization. The slow kinetics and low efficiency of reprogramming methods to generate human induced pluripotent stem cells (iPSCs) impose major limitations on their utility in biomedical applications. Here we describe a chemical approach that dramatically improves (200-fold) the efficiency of iPSC generation from human fibroblasts, within seven days of treatment. This will provide a basis for developing safer, more efficient, nonviral methods for reprogramming human somatic cells.
TL;DR: The SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving to create an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering.
Abstract: We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done.
TL;DR: The multilayered analyses of ChIP-seq and RNA-seq datasets are described, the software packages currently available to perform tasks at each layer are discussed and some upcoming challenges and features for future analysis tools are described.
Abstract: Genome-wide measurements of protein-DNA interactions and transcriptomes are increasingly done by deep DNA sequencing methods (ChIP-seq and RNA-seq). The power and richness of these counting-based measurements comes at the cost of routinely handling tens to hundreds of millions of reads. Whereas early adopters necessarily developed their own custom computer code to analyze the first ChIP-seq and RNA-seq datasets, a new generation of more sophisticated algorithms and software tools are emerging to assist in the analysis phase of these projects. Here we describe the multilayered analyses of ChIP-seq and RNA-seq datasets, discuss the software packages currently available to perform tasks at each layer and describe some upcoming challenges and features for future analysis tools. We also discuss how software choices and uses are affected by specific aspects of the underlying biology and data structure, including genome size, positional clustering of transcription factor binding sites, transcript discovery and expression quantification.
TL;DR: Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two- color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM).
Abstract: The reliance of modern microscopy techniques on photoactivatable fluorescent proteins prompted development of mCherry variants that are initially dark but become red fluorescent after violet-light irradiation. Using ensemble and single-molecule characteristics as selection criteria, we developed PAmCherry1 with excitation/emission maxima at 564/595 nm. Compared to other monomeric red photoactivatable proteins, it has faster maturation, better pH stability, faster photoactivation, higher photoactivation contrast and better photostability. Lack of green fluorescence and single-molecule behavior make monomeric PAmCherry1 a preferred tag for two-color diffraction-limited photoactivation imaging and for super-resolution techniques such as one- and two-color photoactivated localization microscopy (PALM). We performed PALM imaging using PAmCherry1-tagged transferrin receptor expressed alone or with photoactivatable GFP-tagged clathrin light chain. Pair correlation and cluster analyses of the resulting PALM images identified < or =200 nm clusters of transferrin receptor and clathrin light chain at < or =25 nm resolution and confirmed the utility of PAmCherry1 as an intracellular probe.
TL;DR: Mapping large deletions, QTLs and dominant or recessive lethal mutations, as well as identifying additional AT4G35090 mutant alleles, is used for computational analysis of Illumina data.
Abstract: Supplementary Figure 1 Method workflow Supplementary Figure 2 Visual output from SHOREmap DENOVO Supplementary Table 1 Top 10 ranked mutations from the SHOREmap ANNOTATE output Supplementary Table 2 Command line programs, parameters and run time used for the computational analysis of Illumina data Supplementary Table 3 Identification of additional AT4G35090 mutant alleles Supplementary Table 4 Output of SHOREmap ANNOTATE using the interval based on SHOREmap DENOVO data Supplementary Note Mapping large deletions, QTLs and dominant or recessive lethal mutations. Supplementary Methods Lab protocols and computational algorithms Supplementary Data SHORE and SHOREmap example files
TL;DR: This work examines the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis and provides guidance on selection of analysis techniques in the context of a sample workflow.
Abstract: RNA interference (RNAi) has become a powerful technique for reverse genetics and drug discovery, and in both of these areas large-scale high-throughput RNAi screens are commonly performed. The statistical techniques used to analyze these screens are frequently borrowed directly from small-molecule screening; however, small-molecule and RNAi data characteristics differ in meaningful ways. We examine the similarities and differences between RNAi and small-molecule screens, highlighting particular characteristics of RNAi screen data that must be addressed during analysis. Additionally, we provide guidance on selection of analysis techniques in the context of a sample workflow.
TL;DR: A collection of ∼14 million aligned sequence reads from human cell lines has comparable power to detect events as the current generation of DNA microarrays and has over twofold better precision for localizing breakpoints (typically, to within ∼1 kilobase).
Abstract: Cancer results from somatic alterations in key genes, including point mutations, copy-number alterations and structural rearrangements. A powerful way to discover cancer-causing genes is to identify genomic regions that show recurrent copy-number alterations (gains and losses) in tumor genomes. Recent advances in sequencing technologies suggest that massively parallel sequencing may provide a feasible alternative to DNA microarrays for detecting copy-number alterations. Here we present: (i) a statistical analysis of the power to detect copy-number alterations of a given size; (ii) SegSeq, an algorithm to segment equal copy numbers from massively parallel sequence data; and (iii) analysis of experimental data from three matched pairs of tumor and normal cell lines. We show that a collection of approximately 14 million aligned sequence reads from human cell lines has comparable power to detect events as the current generation of DNA microarrays and has over twofold better precision for localizing breakpoints (typically, to within approximately 1 kilobase).
TL;DR: A microfluidic device to trap and properly pair thousands of cells, and observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%.
Abstract: Cell fusion has been used for many different purposes, including generation of hybridomas and reprogramming of somatic cells. The fusion step is the key event in initiation of these procedures. Standard fusion techniques, however, provide poor and random cell contact, leading to low yields. We present here a microfluidic device to trap and properly pair thousands of cells. Using this device, we paired different cell types, including fibroblasts, mouse embryonic stem cells and myeloma cells, achieving pairing efficiencies up to 70%. The device is compatible with both chemical and electrical fusion protocols. We observed that electrical fusion was more efficient than chemical fusion, with membrane reorganization efficiencies of up to 89%. We achieved greater than 50% properly paired and fused cells over the entire device, fivefold greater than with a commercial electrofusion chamber and observed reprogramming in hybrids between mouse embryonic stem cells and mouse embryonic fibroblasts.
TL;DR: A new generation of methods are being developed to tackle the challenges of short reads, while taking advantage of the high coverage the new sequencing technologies provide.
Abstract: In the last several years, a number of studies have described large-scale structural variation in several genomes. Traditionally, such methods have used whole-genome array comparative genome hybridization or single-nucleotide polymorphism arrays to detect large regions subject to copy-number variation. Later techniques have been based on paired-end mapping of Sanger sequencing data, providing better resolution and accuracy. With the advent of next-generation sequencing, a new generation of methods is being developed to tackle the challenges of short reads, while taking advantage of the high coverage the new sequencing technologies provide. In this survey, we describe these methods, including their strengths and their limitations, and future research directions.
TL;DR: An EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the ∼10-nm localization precision of its parent is reported.
Abstract: Photoconvertible fluorescent proteins are potential tools for investigating dynamic processes in living cells and for emerging super-resolution microscopy techniques. Unfortunately, most probes in this class are hampered by oligomerization, small photon budgets or poor photostability. Here we report an EosFP variant that functions well in a broad range of protein fusions for dynamic investigations, exhibits high photostability and preserves the approximately 10-nm localization precision of its parent.
TL;DR: Phymm, a classifier for metagenomic data, is presented that has been trained on 539 complete, curated genomes and can accurately classify reads as short as 100 base pairs, a substantial improvement over previous composition-based classification methods.
Abstract: This algorithm for the assignment of phylogenetic groups to fragments generated by metagenomic sequencing projects improves on the currently required 1 kb fragment length for classification. Trained on 539 complete genomes, Phymm can classify reads as short as 100 bp. Combining Phymm with the sequence alignment algorithm BLAST further improves accuracy.
TL;DR: A fluorescent sensor of adenylate nucleotides is constructed by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii, and can be used to monitor the ATP:ADP ratio during live-cell imaging.
Abstract: We constructed a fluorescent sensor of adenylate nucleotides by combining a circularly permuted variant of GFP with a bacterial regulatory protein, GlnK1, from Methanococcus jannaschii. The sensor's affinity for Mg-ATP was <100 nM, as seen for other members of the bacterial PII regulator family, a surprisingly high affinity given that normal intracellular ATP concentration is in the millimolar range. ADP bound the same site of the sensor as Mg-ATP, competing with it, but produced a smaller change in fluorescence. At physiological ATP and ADP concentrations, the binding site is saturated, but competition between the two substrates causes the sensor to behave as a nearly ideal reporter of the ATP:ADP concentration ratio. This principle for sensing the ratio of two analytes by competition at a high-affinity site probably underlies the normal functioning of PII regulatory proteins. The engineered sensor, Perceval, can be used to monitor the ATP:ADP ratio during live-cell imaging.