Brief Communications in 2009

A combination of static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy is used for the in vivo analysis of receptor-ligand interactions in living zebrafish embryos. The measured binding affinities suggest that Fgf8 binds to both Fgfr1 and Fgfr4 during gastrulation.

This technique, adapted from mosaic analysis with double markers in mice, relies on mitotic recombination to reconstitute sequences encoding EGFP or mRFP1. After cell division, each daughter cell contains one fluorescent marker, causing a green and a red twin spot that can be traced through development.

A Gal4-based system in Drosophila reports on gene expression at a given developmental stage combined with lineage information on expression at earlier developmental stages.

Nucleotide analogs modified with a free 3′ hydroxyl, maintaining the interactions at the polymerase active site, and a cleavable linker, attaching a fluorescent dye and an inhibitor, are efficient at reading homopolymer runs in a single-molecule sequencing reaction.

Multistage mass spectrometry and algorithms for spectral alignment and dereplication allow sequencing of nonribosomal peptides, pharmacologically important compounds that are not encoded in the genome but built by nonribosomal peptide synthetases.

This array-based discovery tool creates linkage between functional mutations and selectable markers across a bacterial genome and can thus distinguish between adaptive and neutral mutations.

Iterative cycles of metabolic modeling and experimental open reading frame verification in Chlamydomonas reinhardtii lay the groundwork for more accurate gene annotation and provide resources for metabolic engineering.

A generally applicable approach to analyze intact membrane protein complexes by mass spectrometry is reported. This method allows subunit stoichiometry, lipid binding and the effects of post-translational modifications on complex formation to be explored.

A system for inducible protein degradation, originally developed for mammalian cells, is applied to essential viral proteins and will allow functional studies in a wide range of viruses.

Combinations of fluorescently labeled peptide–major histocompatability (pMHC) tetramers are used to simultaneously detect T cells with multiple antigen specificities from human blood samples. Also in this issue, Hadrup et al. present a very similar combinatorial encoding approach.

Concatenated PCR products serve as subgenomic traps in this targeted genome capture technique; subsequent high-throughput sequencing allows the detection of nucleotide and structural variations in the captured genomic regions.

There have been many attempts to measure gene expression in single cells but counting several different mRNAs in the same cell has been a challenge. A reusable single-cell cDNA library immobilized on beads allows quantitative measurement of multiple mRNAs in a single cell with a large dynamic range and small experimental error.

Mouse embryonic stem cell lines from the C57BL/6 strain are reported. The lines are highly germline competent, suitable for high-throughput genetic manipulation and will enable the generation of large knockout mouse resources.

A combination of gradient refractive index lenses with plano-convex lenses produces high-resolution microlenses with image quality similar to a conventional high quality microscope objective. The microlenses are capable of imaging dendritic spines on hippocampal neurons in live mice.

Using a topographically patterned substrate for immobilization of single yeast cells and a piezo-impact micromanipulator to transiently disrupt the cell wall, molecules can be physically introduced into yeast.

The protein interaction platform or PIP assay uses a viral scaffolding protein fused to a bait and a fluorescent reporter protein fused to putative prey as the basis for a simple visual screen for protein-protein interactions in yeast.

Genomic fosmid libraries for Drosophila melanogaster and Drosophila pseudoobscura with an average insert size of 36 kilobases can easily be tagged and inserted into the fly genome. These resources will be valuable for evolutionary and developmental studies in the fly.

Two bacterial artificial chromosome (BAC) libraries, spanning almost the entire D. melanogaster genome in insert sizes of 20 and 80 kb, that allow easy integration into the fruit fly genome at defined docking sites provide a rich resource to study gene expression and function.

On-array synthesis of over 20,000 shRNAs at a coverage of ∼30 shRNAs per gene, followed by cloning into lentiviral shRNA libraries and deconvolution of the complex libraries by deep sequencing, ensures high confidence in the observed knockdown phenotypes with low false-negative rates and few off-target hits.

Expressing uracil phosphoribosyltransferase in specific tissues in the fly allows the incorporation of 4-thiouracil into newly synthesized RNA in vivo. The thio-labeled RNA can then be isolated and analyzed by routine procedures allowing the cell type–specific measure of RNA synthesis and decay rates.