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Nature Methods

• A proteome chip approach reveals new DNA damage recognition activities in Escherichia coli

Chien-Sheng Chen1,2,4, Ekaterina Korobkova3,4, Hao Chen3, Jian Zhu1, Xing Jian3, Sheng-Ce Tao1, Chuan He3 & Heng Zhu1

Despite the fact that many genomes have been decoded, proteome chips comprising individually purified proteins have been reported only for budding yeast, mainly because of the complexity and difficulty of high-throughput protein purification. To facilitate proteomics studies in prokaryotes, we have developed a high-throughput protein purification protocol that allowed us to purify 4,256 proteins encoded by the Escherichia coli K12 strain within 10 h. The purified proteins were then spotted onto glass slides to create E. coli proteome chips. We used these chips to develop assays for identifying proteins involved in the recognition of potential base damage in DNA. By using a group of DNA probes, each containing a mismatched base pair or an abasic site, we found a small number of proteins that could recognize each type of probe with high affinity and specificity. We further evaluated two of these proteins, YbaZ and YbcN, by biochemical analyses. The assembly of libraries containing DNA probes with specific modifications and the availability of E. coli proteome chips have the potential to reveal important interactions between proteins and nucleic acids that are time-consuming and difficult to detect using other techniques.

NATURE METHODS | VOL.5 NO.1 | JANUARY 2008 | 69

• Highly inclined thin illumination enables clear single-molecule imaging in cells

Makio Tokunaga1–3, Naoko Imamoto4,5 & Kumiko Sakata-Sogawa2

We describe a simple illumination method of fluorescence microscopy for molecular imaging. Illumination by a highly inclined and thin beam increases image intensity and decreases background intensity, yielding a signal/background ratio about eightfold greater than that of epi-illumination. A high ratio yielded clear single-molecule images and three-dimensional images using cultured mammalian cells, enabling one to visualize and quantify molecular dynamics, interactions and kinetics in cells for molecular systems biology.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 159

• An activity-independent selection system of thermostable protein variants

He´le`ne Chautard1, Emilio Blas-Galindo2, Thierry Menguy1, Laure Grand’Moursel1, Felipe Cava2, Jose´ Berenguer2 & Marc Delcourt1

We describe an activity-independent method for the selection of thermostable mutants of any protein. It is based on a fusion construct comprising the protein of interest and a thermostable antibiotic resistance reporter, in such a way that thermostable mutants provide increased resistance in a thermophile. We isolated thermostable mutants of three human interferons and of two enzymes to demonstrate the applicability of the system.

NATURE METHODS | VOL.4 NO.11 | NOVEMBER 2007 | 919

• In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Jose´ Luis Royo1,2,4, Pablo Daniel Becker2,4, Eva Marı´a Camacho1, Angel Cebolla3, Claudia Link2, Eduardo Santero1 & Carlos Alberto Guzma´n2

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20–150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine–converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.

NATURE METHODS | VOL.4 NO.11 | NOVEMBER 2007 | 937

• Gene synthesis by circular assembly amplification

Duhee Bang & George M Church

Here we report the development of a gene-synthesis technology, circular assembly amplification. In this approach, we first constructed exonuclease-resistant circular DNA via simultaneous ligation of oligonucleotides. Exonuclease- and subsequent mismatch cleaving endonuclease–mediated degradation of the resulting ligation mixture eliminated error-rich products, thereby substantially improving gene-synthesis quality. We used this method to construct genes encoding a small thermostable DNA polymerase, a highly repetitive DNA sequence and large (44 kb) constructs.

NATURE METHODS | VOL.5 NO.1 | JANUARY 2008 | 37

• Epitope tagging of endogenous proteins for genome-wide ChIP-chip studies

Xiaodong Zhang1,7, Chunguang Guo2,7, Yueting Chen1,7, Hennady P Shulha3, Michael P Schnetz1, Thomas LaFramboise1, Cynthia F Bartels1, Sanford Markowitz4, Zhiping Weng3,5, Peter C Scacheri1 & Zhenghe Wang1,6

We developed a strategy to introduce epitope tag–encoding DNA into endogenous loci by homologous recombination–mediated ‘knock-in’. The tagging method is straightforward, can be applied to many loci and several human somatic cell lines, and can facilitate many functional analyses including western blot, immunoprecipitation, immunofluorescence and chromatin immunoprecipitation–microarray (ChIP-chip). The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 163

• Plasmid-chromosome shuffling for non-deletion alleles in yeast

Zhiwei Huang1,4, Richard S Sucgang1,4, Yu-yi Lin3, Xiaomin Shi1, Jef D Boeke3 & Xuewen Pan1,2

Here we describe a facile plasmid-chromosome shuffling technique for generating and analyzing non-deletion alleles in the yeast Saccharomyces cerevisiae. This technique takes advantage of an existing set of genome-wide haploid-convertible heterozygous diploid yeast knockout mutants. This simple method will facilitate characterization of essential gene functions and genome-wide investigation of protein structure-function relationships.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 167

• High-density mapping of single-molecule trajectories with photoactivated localization microscopy

Suliana Manley1, Jennifer M Gillette1, George H Patterson1, Hari Shroff2, Harald F Hess2, Eric Betzig2 & Jennifer Lippincott-Schwartz1

We combined photoactivated localization microscopy (PALM) with live-cell single-particle tracking to create a new method termed sptPALM. We created spatially resolved maps of single-molecule motions by imaging the membrane proteins Gag and VSVG, and obtained several orders of magnitude more trajectories per cell than traditional single-particle tracking enables. By probing distinct subsets of molecules, sptPALM can provide insight into the origins of spatial and temporal heterogeneities in membranes.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 155

• Printing protein arrays from DNA arrays

Mingyue He1,4, Oda Stoevesandt1,3,4, Elizabeth A Palmer1,3, Farid Khan1,3, Olle Ericsson2 & Michael J Taussig1,3

We describe a method, DNA array to protein array (DAPA), which allows the ‘printing’ of replicate protein arrays directly from a DNA array template using cell-free protein synthesis. At least 20 copies of a protein array can be obtained from a single DNA array. DAPA eliminates the need for separate protein expression, purification and spotting, and also overcomes the problem of long-term functional storage of surface-bound proteins.

NATURE METHODS | VOL.5 NO.2 | FEBRUARY 2008 | 175

• A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiae

Grace Marie Jones1, Jim Stalker2, Sean Humphray2, Anthony West2, Tony Cox2, Jane Rogers2, Ian Dunham2 & Gregory Prelich1

Modern genetic analysis requires the development of new resources to systematically explore gene function in vivo. Overexpression screens are a powerful method to investigate genetic pathways, but the goal of routine and comprehensive overexpression screens has been hampered by the lack of systematic libraries. Here we describe the construction of a systematic collection of the Saccharomyces cerevisiae genome in a high-copy vector and its validation in two overexpression screens.

NATURE METHODS | VOL.5 NO.3 | MARCH 2008 | 239

• A systematic library for comprehensive overexpression screens in Saccharomyces cerevisiaeHomogeneous reporter system enables

quantitative functional assessment of multiple transcription factors

Sergei Romanov1, Alexander Medvedev1, Maria Gambarian1, Natalia Poltoratskaya1, Matt Moeser1, Liubov Medvedeva1, Mikhail Gambarian2, Luda Diatchenko3 & Sergei Makarov1

We developed a high-content reporter system that allows quantitative assessment of activities of multiple transcription factors (TFs) in a eukaryotic cell. The system comprises a library of reporter constructs that are evaluated according to their transcription rates. All reporters produce essentially identical messages that are subjected to ‘processing’, which generates a spectrum of distinguishable fragments that are analyzed quantitatively. The homogeneity of the reporter library afforded inherently uniform detection conditions for all reporters and provided repeatability, accuracy and robustness of assessment. We showed that this technology can be used to identify pathways transmitting cell responses to inducers, and that the profile of TF activities generated using this system represents a stable and sustained cell signature that clearly distinguishes different cell types and pathological conditions. This technology provides a framework for functional characterization of signal transduction networks through profiling activities of multiple TFs.

NATURE METHODS | VOL.5 NO.3 | MARCH 2008 | 253

• MicroRNA quantitation from a single cell by PCR using SYBR® Green detection and LNA-based primers

We describe a new, highly sensitive and specific PCR approach for quantitation of microRNAs (miRNAs): the miRCURY™ LNA microRNA PCR system. The method, which allows detection of 10 copies of miRNA, is enabled by the use of Locked Nucleic Acids (LNA™). The LNA-conferred sensitivity facilitates accurate detection of miRNA expression in a single cell.

NATURE METHODS | FEBRUARY 2008 | iii ADVERTISING FEATURE/EXIQON