Promoter nucleosome organization shapes the evolution of gene expression

Understanding why genes evolve at different rates is fundamental to evolutionary thinking. In species of the budding yeast, the rate at which genes diverge in expression correlates with the organization of their promoter nucleosomes: genes lacking a nucleosome-free region (denoted OPN for "Occupied Proximal Nucleosomes") vary widely between the species, while the expression of those containing NFR (denoted DPN for "Depleted Proximal Nucleosomes") remains largely conserved. To examine if early evolutionary dynamics contributes to this difference in divergence, we artificially selected for high expression of GFP-fused proteins. Surprisingly, selection was equally successful for OPN and DPN genes, with -80% of genes in each group stably increasing in expression by a similar amount. Notably, the two groups adapted by distinct mechanisms: DPN-selected strains duplicated large genomic regions, while OPN-selected strains favored trans mutations not involving duplications. When selection was removed, DPN (but not OPN) genes reverted rapidly to wild-type expression levels, consistent with their lower diversity between species. Our results suggest that promoter organization constrains the early evolutionary dynamics and in this way biases the path of long-term evolution.     

Using bleach-chase to measure protein half-lives in living cells

Protein removal has a central role in numerous cellular processes. Obtaining systematic measurements of multiple protein removal rates is necessary to understand the principles that govern these processes, but it is currently a major technical challenge. To address this, we developed 'bleach-chase', a noninvasive method for measuring the half-lives of multiple proteins at high temporal resolution in living cells. The method uses a library of annotated human reporter cell clones, each with a unique fluorescently tagged protein expressed from its native chromosomal location. In this protocol, we detail a simple procedure that bleaches the cells and uses time-lapse fluorescence microscopy and automated image analysis to systematically measure the half-life dynamics of multiple proteins. The duration of the protocol is 4-5 d. The method may be applicable to a wide range of fluorescently tagged proteins and cell lines.     

Crystal structure of human mitochondrial PheRS complexed with tRNA(Phe) in the active "open" state

Monomeric human mitochondrial phenylalanyl-tRNA synthetase (PheRS), or hmPheRS, is the smallest known enzyme exhibiting aminoacylation activity. HmPheRS consists of only two structural domains and differs markedly from heterodimeric eukaryotic cytosolic and bacterial analogs both in the domain organization and in the mode of tRNA binding. Here, we describe the first crystal structure of mitochondrial aminoacyl-tRNA synthetase (aaRS) complexed with tRNA at a resolution of 3.0 Å. Unlike bacterial PheRSs, the hmPheRS recognizes C74, the G1–C72 base pair, and the “discriminator” base A73, proposed to contribute to tRNA^Phe identity in the yeast mitochondrial enzyme. An interaction of the tRNA acceptor stem with the signature motif 2 residues of hmPheRS is of critical importance for the stabilization of the CCA-extended conformation and its correct placement in the synthetic site of the enzyme. The crystal structure of hmPheRS–tRNA^Phe provides direct evidence that the formation of the complex with tRNA requires a significant rearrangement of the anticodon-binding domain from the “closed” to the productive “open” state. Global repositioning of the domain is tRNA modulated and governed by long-range electrostatic interactions.     

Intracellular Mechanics and Activity of Breast Cancer Cells Correlate with Metastatic Potential

Mechanics of cancer cells are directly linked to their metastatic potential, or ability to produce a secondary tumor at a distant site. Metastatic cells survive in the circulatory system in a non-adherent state, and can squeeze through barriers in the body. Such considerable structural changes in cells rely on rapid remodeling of internal structure and mechanics. While external mechanical measurements have demonstrated enhanced pliability of cancer cells with increased metastatic potential, little is known about dynamics of their interior and we expect that to change significantly in metastatic cells. We perform a comparative study, using particle-tracking to evaluate the intracellular mechanics of living epithelial breast cells with varying invasiveness. Particles in all examined cell lines exhibit super-diffusion with a scaling exponent of 1.4 at short lag times, likely related to active transport by fluctuating microtubules and their associated molecular motors. Specifics of probe-particle transport differ between the cell types, depending on the cytoskeleton network-structure and interactions with it. Our study shows that the internal microenvironment of the highly metastatic cells evaluated here is more pliable and their cytoskeleton is less dense than the poorly metastatic and benign cells. We thus reveal intracellular structure and mechanics that can support the unique function and invasive capabilities of highly metastatic cells.     

Expression,purification, and isotope labeling of the Fv of the human HIV-1 neutralizing antibody 447-52D for NMR studies

The Fv is the smallest antigen binding fragment of the antibody and is made of the variable domains of the light and heavy chains, V(L) and V(H), respectively. The 26-kDa Fv is amenable for structure determination in solution using multi-dimensional hetero-nuclear NMR spectroscopy. The human monoclonal antibody 447-52D neutralizes a broad spectrum of HIV-1 isolates. This anti-HIV-1 antibody elicited in an infected patient is directed against the third variable loop (V3) of the envelope glycoprotein (gp120) of the virus. The V3 loop is an immunodominant neutralizing epitope of HIV-1. To obtain the 447-52D Fv for NMR studies, an Escherichia coli bicistronic expression vector for the heterodimeric 447-52D Fv and vectors for single chain Fv and individually expressed V(H) and V(L) were constructed. A pelB signal peptide was linked to the antibody genes to enable secretion of the expressed polypeptides into the periplasm. For easy cloning of any antibody gene without potential modification of the antibody sequence, restriction sites were introduced in the pelB sequence and following the termination codon. A set of oligonucleotides that prime the leader peptide genes of all potential antibody human antibodies were designed as backward primers. The forward primers for the V(L) and V(H) were based on constant region sequences. The 447-52D Fv could not be expressed either by a bicistronic vector or as single chain Fv, probably due to its toxicity to Escherichia coli. High level of expression was obtained by individual expression of the V(H) and the V(L) chains, which were then purified and recombined to generate a soluble and active 447-52D Fv fragment. The V(L) of mAb 447-52D was uniformly labeled with 13C and 15N nuclei (U-13C/15N). Preliminary NMR spectra demonstrate that structure determination of the recombinant 447-52D Fv and its complex with V3 peptides is feasible.     

Advanced computational techniques for re-sequencing DNA with polymerase signaling assay arrays

Re-sequencing, the identification of the specific variants in a sequence of interest compared with a known genomic sequence, is a ubiquitous task in today’s biology. Universal arrays, which interrogate all possible oligonucleotides of a certain length in a target sequence, have been suggested for computationally determining a polynucleotide sequence from its oligonucleotide content. We present here new methods that use such arrays for re-sequencing. Our methods are applied to data obtained by the polymerase signaling assay, which arrays single-based primer extension reactions for either universal or partial arrays of pentanucleotides. The computational analysis uses the spectrum alignment algorithm, which is refined and enhanced here in order to overcome noise incurred by the use of such short primers. We present accurate re-sequencing results for both synthetic and amplified DNA molecules.     

Clustering and conservation patterns of human microRNAs

MicroRNAs (miRNAs) are ~22 nt-long non-coding RNA molecules, believed to play important roles in gene regulation. We present a comprehensive analysis of the conservation and clustering patterns of known miRNAs in human. We show that human miRNA gene clustering is significantly higher than expected at random. A total of 37% of the known human miRNA genes analyzed in this study appear in clusters of two or more with pairwise chromosomal distances of at most 3000 nt. Comparison of the miRNA sequences with their homologs in four other organisms reveals a typical conservation pattern, persistent throughout the clusters. Furthermore, we show enrichment in the typical conservation patterns and other miRNA-like properties in the vicinity of known miRNA genes, compared with random genomic regions. This may imply that additional, yet unknown, miRNAs reside in these regions, consistent with the current recognition that there are overlooked miRNAs. Indeed, by comparing our predictions with cloning results and with identified miRNA genes in other mammals, we corroborate the predictions of 18 additional human miRNA genes in the vicinity of the previously known ones. Our study raises the proportion of clustered human miRNAs that are <3000 nt apart to 42%. This suggests that the clustering of miRNA genes is higher than currently acknowledged, alluding to its evolutionary and functional implications.