Transcription of highly expressed genes has been shown to occur in stochastic bursts. But the origin of such ubiquitous phenomenon has not been understood. Here, we present the mechanism in bacteria. ...We developed a high-throughput, in vitro, single-molecule assay to follow transcription on individual DNA templates in real time. We showed that positive supercoiling buildup on a DNA segment by transcription slows down transcription elongation and eventually stops transcription initiation. Transcription can be resumed upon gyrase binding to the DNA segment. Furthermore, using single-cell mRNA counting fluorescence in situ hybridization (FISH), we found that duty cycles of transcriptional bursting depend on the intracellular gyrase concentration. Together, these findings prove that transcriptional bursting of highly expressed genes in bacteria is primarily caused by reversible gyrase dissociation from and rebinding to a DNA segment, changing the supercoiling level of the segment.
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•A high-throughput single-molecule assay follows in vitro transcription in real time•Positive supercoiling buildup inhibits transcription initiation and elongation•Transcriptional bursting is dependent on the intracellular gyrase level•Gyrase dissociation from and rebinding to a DNA loop cause transcriptional bursts
Transcriptional bursting in bacteria is inhibited by positive supercoiling of DNA, and the extent of bursting is consequently regulated by the levels of intracellular gyrase, an enzyme that releases positive supercoiling.
Kindred cells can have different genomes because of dynamic changes in DNA. Single-cell sequencing is needed to characterize these genomic differences but has been hindered by whole-genome ...amplification bias, resulting in low genome coverage. Here, we report on a new amplification method—multiple annealing and looping-based amplification cycles (MALBAC)—that offers high uniformity across the genome. Sequencing MALBAC-amplified DNA achieves 93% genome coverage ≥1x for a single human cell at 25x mean sequencing depth. We detected digitized copy-number variations (CNVs) of a single cancer cell. By sequencing three kindred cells, we were able to identify individual single-nucleotide variations (SNVs), with no false positives detected. We directly measured the genome-wide mutation rate of a cancer cell line and found that purine-pyrimidine exchanges occurred unusually frequently among the newly acquired SNVs.
Three-dimensional genome structures play a key role in gene regulation and cell functions. Characterization of genome structures necessitates single-cell measurements. This has been achieved for ...haploid cells but has remained a challenge for diploid cells. We developed a single-cell chromatin conformation capture method, termed Dip-C, that combines a transposon-based whole-genome amplification method to detect many chromatin contacts, called META (multiplex end-tagging amplification), and an algorithm to impute the two chromosome haplotypes linked by each contact. We reconstructed the genome structures of single diploid human cells from a lymphoblastoid cell line and from primary blood cells with high spatial resolution, locating specific single-nucleotide and copy number variations in the nucleus. The two alleles of imprinted loci and the two X chromosomes were structurally different. Cells of different types displayed statistically distinct genome structures. Such structural cell typing is crucial for understanding cell functions.
Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules often exist in low copy numbers and are difficult to detect in single ...cells. We carried out quantitative system-wide analyses of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell's protein and mRNA copy numbers for any given gene are uncorrelated.
We investigate the 3D quantum Hall effect in Weyl semimetals and elucidate a global picture of the edge states. The edge states hosting 3D quantum Hall effect are combinations of Fermi arcs and ...chiral Landau bands dispersing along the magnetic field direction. The Hall conductance, σxzH see Fig. 4, shows quantized plateaus with the variance of the magnetic field when the Fermi level is at the Weyl node. However, the chiral Landau bands can change the spatial distribution of the edge states, especially under a tilted magnetic field, and the resulting edge states lead to distinctive Hall transport phenomena. A tilted magnetic field contributes an intrinsic value to σxzH and such an intrinsic value is determined by the tilting angle θ between the magnetic field and the y axis see Fig. 1(c). Particularly, even if the perpendicular magnetic field is fixed, σxzH will change its sign with an abrupt spatial shift of the edge states when θ exceeds a critical angle θc. Our work uncovers the novel edge-state nature of the 3D quantum Hall effect in Weyl semimetals.
Vibrational spectroscopy has been extensively applied to the study of molecules in gas phase, in condensed phase, and at interfaces. The transition from spectroscopy to spectroscopic imaging of ...living systems, which allows the spectrum of biomolecules to act as natural contrast, is opening new opportunities to reveal cellular machinery and to enable molecule-based diagnosis. Such a transition, however, involves more than a simple combination of spectrometry and microscopy. We review recent efforts that have pushed the boundary of the vibrational spectroscopic imaging field in terms of spectral acquisition speed, detection sensitivity, spatial resolution, and imaging depth. We further highlight recent applications in functional analysis of single cells and in label-free detection of diseases.
Bacterial chromosomes are confined in submicrometer-sized nucleoids. Chromosome organization is facilitated by nucleoid-associated proteins (NAPs), but the mechanisms of action remain elusive. In ...this work, we used super-resolution fluorescence microscopy, in combination with a chromosome-conformation capture assay, to study the distributions of major NAPs in live Escherichia coli cells. Four NAPs—HU, Fis, IHF, and StpA—were largely scattered throughout the nucleoid. In contrast, H-NS, a global transcriptional silencer, formed two compact clusters per chromosome, driven by oligomerization of DNA-bound H-NS through interactions mediated by the amino-terminal domain of the protein. H-NS sequestered the regulated operons into these clusters and juxtaposed numerous DNA segments broadly distributed throughout the chromosome. Deleting H-NS led to substantial chromosome reorganization. These observations demonstrate that H-NS plays a key role in global chromosome organization in bacteria.
Single-cell genomics is important for biology and medicine. However, current whole-genome amplification (WGA) methods are limited by low accuracy of copy-number variation (CNV) detection and low ...amplification fidelity. Here we report an improved single-cell WGA method, Linear Amplification via Transposon Insertion (LIANTI), which outperforms existing methods, enabling micro-CNV detection with kilobase resolution. This allowed direct observation of stochastic firing of DNA replication origins, which differs from cell to cell. We also show that the predominant cytosine-to-thymine mutations observed in single-cell genomics often arise from the artifact of cytosine deamination upon cell lysis. However, identifying single-nucleotide variations (SNVs) can be accomplished by sequencing kindred cells. We determined the spectrum of SNVs in a single human cell after ultraviolet radiation, revealing their nonrandom genome-wide distribution.
Sensory neurons in the mouse eye and nose have unusual chromatin organization. Here we report their three-dimensional (3D) genome structure at 20-kilobase (kb) resolution, achieved by applying our ...recently developed diploid chromatin conformation capture (Dip-C) method to 409 single cells from the retina and the main olfactory epithelium of adult and newborn mice. The 3D genome of rod photoreceptors exhibited inverted radial distribution of euchromatin and heterochromatin compared with that of other cell types, whose nuclear periphery is mainly heterochromatin. Such genome-wide inversion is not observed in olfactory sensory neurons (OSNs). However, OSNs exhibited an interior bias for olfactory receptor (OR) genes and enhancers, in clear contrast to non-neuronal cells. Each OSN harbored multiple aggregates of OR genes and enhancers from different chromosomes. We also observed structural heterogeneity of the protocadherin gene cluster. This type of genome organization may provide the structural basis of the 'one-neuron, one-receptor' rule of olfaction.
The Goos-Hänchen (GH) shift and the Imbert-Fedorov (IF) shift are optical phenomena which describe the longitudinal and transverse lateral shifts at the reflection interface, respectively. Here, we ...predict the GH and IF shifts in Weyl semimetals (WSMs)-a promising material harboring low energy Weyl fermions, a massless fermionic cousin of photons. Our results show that the GH shift in WSMs is valley independent, which is analogous to that discovered in a 2D relativistic material-graphene. However, the IF shift has never been explored in nonoptical systems, and here we show that it is valley dependent. Furthermore, we find that the IF shift actually originates from the topological effect of the system. Experimentally, the topological IF shift can be utilized to characterize the Weyl semimetals, design valleytronic devices of high efficiency, and measure the Berry curvature.
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