Effects of molecular memory and bursting on fluctuations in gene expression

Many cellular components are present in such low numbers per cell that random births and deaths of individual molecules can cause substantial "noise" in concentrations. But biochemical events do not necessarily occur in single steps of individual molecules. Some processes are greatly randomized when synthesis or degradation occurs in large bursts of many molecules during a short time interval. Conversely, each birth or death of a macromolecule could involve several small steps, creating a memory between individual events. We present a generalized theory for stochastic gene expression, formulating the variance in protein abundance in terms of the randomness of the individual gene expression events. We show that common types of molecular mechanisms can produce gestation and senescence periods that reduce noise without requiring higher abundances, shorter lifetimes, or any concentration-dependent control loops. We also show that most single-cell experimental methods cannot distinguish between qualitatively different stochastic principles, although this in turn makes such methods better suited for identifying which components introduce fluctuations. Characterizing the random events that give rise to noise in concentrations instead requires dynamic measurements with single-molecule resolution.     

Counting low-copy number proteins in a single cell

We have designed a microfluidic device in which we can manipulate, lyse, label, separate, and quantify the protein contents of a single cell using single-molecule fluorescence counting. Generic labeling of proteins is achieved through fluorescent-antibody binding. The use of cylindrical optics enables high-efficiency (approximately 60%) counting of molecules in micrometer-sized channels. We used this microfluidic device to quantify beta2 adrenergic receptors expressed in insect cells (SF9). We also analyzed phycobiliprotein contents in individual cyanobacterial cells (Synechococcus sp. PCC 7942) and observed marked differences in the levels of specific complexes in cell populations that were grown under nitrogen-depleted conditions.     

Polymerase exchange during Okazaki fragment synthesis observed in living cells

DNA replication machineries have been studied extensively, but the kinetics of action of their components remains largely unknown. We report a study of DNA synthesis during replication in living Escherichia coli cells. Using single-molecule microscopy, we observed repetitive fluorescence bursts of single polymerase IIIs (Pol IIIs), indicating polymerase exchange at the replication fork. Fluctuations in the amount of DNA-bound single-stranded DNA-binding protein (SSB) reflect different speeds for the leading- and lagging-strand DNA polymerases. Coincidence analyses of Pol III and SSB fluctuations show that they correspond to the lagging-strand synthesis and suggest the use of a new Pol III for each Okazaki fragment. Based on exchanges involving two Pol IIIs, we propose that the third polymerase in the replisome is involved in lagging-strand synthesis.     

A stochastic single-molecule event triggers phenotype switching of a bacterial cell

By monitoring fluorescently labeled lactose permease with single-molecule sensitivity, we investigated the molecular mechanism of how an Escherichia coli cell with the lac operon switches from one phenotype to another. At intermediate inducer concentrations, a population of genetically identical cells exhibits two phenotypes: induced cells with highly fluorescent membranes and uninduced cells with a small number of membrane-bound permeases. We found that this basal-level expression results from partial dissociation of the tetrameric lactose repressor from one of its operators on looped DNA. In contrast, infrequent events of complete dissociation of the repressor from DNA result in large bursts of permease expression that trigger induction of the lac operon. Hence, a stochastic single-molecule event determines a cell's phenotype.     

The complex folding network of single calmodulin molecules

Direct observation of the detailed conformational fluctuations of a single protein molecule en route to its folded state has so far been realized only in silico. We have used single-molecule force spectroscopy to study the folding transitions of single calmodulin molecules. High-resolution optical tweezers assays in combination with hidden Markov analysis reveal a complex network of on- and off-pathway intermediates. Cooperative and anticooperative interactions across domain boundaries can be observed directly. The folding network involves four intermediates. Two off-pathway intermediates exhibit non-native interdomain interactions and compete with the ultrafast productive folding pathway.     

串联型黄色荧光蛋白在Vero细胞中的表达

试验旨在探索串联型黄色荧光蛋白(YFP)在真核细胞中的表达,以便于构建用于球虫转基因研究的载体.用PCR方法克隆了不含鼠鸟氨酸脱羧酶PEST序列的目的DNA片段YFP-1和YFP-2,并将其依次连接于原始载体pd2EYFP-N1以替换d2EYFP报告基因,将重组载体电转染Vero细胞,观察荧光情况.结果显示,重组载体pYFP-YFP-N1能够在Vero细胞中正常表达,串联型YFP基因可用于球虫转基因中载体构建.     

Fluorescence spectroscopy of single biomolecules

Recent advances in single-molecule detection and single-molecule spectroscopy at room temperature by laser-induced fluorescence offer new tools for the study of individual macromolecules under physiological conditions. These tools relay conformational states, conformational dynamics, and activity of single biological molecules to physical observables, unmasked by ensemble averaging. Distributions and time trajectories of these observables can therefore be measured during a reaction without the impossible need to synchronize all the molecules in the ensemble. The progress in applying these tools to biological studies with the use of fluorophores that are site-specifically attached to macromolecules is reviewed.     

借助纳米珠制作单分子DNA芯片

传统的单分子DNA芯片制作技术主要依靠稀溶液铺设,有很大的随机性,即有许多DNA分子重叠而无法观察,还有一部分基片上没有样品,造成浪费,为克服上述缺点,作者采用了一种新的单分子芯片铺设技术,纳米珠作为单分子DNA的连接载体,利用其空间位阻作用将DNA分子沉降到经过表面化学处理的基片表面,纳米珠的直径和DNA长度控制芯片上样点间距,去除纳米珠后,获得单分子DNA纳米阵列芯片。为单分子、高通量DNA芯片的制作做了新的探索。这个技术的完善将为今后单分子DNA研究开辟新的途径。     

Zero-mode waveguides for single-molecule analysis at high concentrations

Optical approaches for observing the dynamics of single molecules have required pico- to nanomolar concentrations of fluorophore in order to isolate individual molecules. However, many biologically relevant processes occur at micromolar ligand concentrations, necessitating a reduction in the conventional observation volume by three orders of magnitude. We show that arrays of zero-mode waveguides consisting of subwavelength holes in a metal film provide a simple and highly parallel means for studying single-molecule dynamics at micromolar concentrations with microsecond temporal resolution. We present observations of DNA polymerase activity as an example of the effectiveness of zero-mode waveguides for performing single-molecule experiments at high concentrations.     

CMV启动子与艾美耳球虫基因侧翼序列对黄色荧光蛋白表达调控作用的研究

探索CMV启动子与艾美耳球虫基因侧翼序列对串联型黄色荧光蛋白(YFP)表达的调控作用,将含有串联型YFP、CMV启动子、柔嫩艾美耳球虫组蛋白4(Histone4)上游启动序列和肌动蛋白(Actin)下游调控序列的重组载体pCMV-YFP-YFP、pH4-YFP-YFP-ACT电转染Vero细胞和柔嫩艾美耳球虫子孢子,观察荧光情况.结果证明,CMV启动子不能驱动YFP在柔嫩艾美耳球虫中正常表达,而其自身基因的侧翼序列能够启动和调控串联型YFP在柔嫩艾美耳球虫中的正常表达.     

水稻条纹病毒编码的NS2蛋白的亚细胞定位分析

通过RT-PCR克隆获得水稻条纹病毒（RSV）编码的蛋白NS2和拟南芥柯浩体蛋白Atcoilin的cDNA,将两者分别与表达载体pEarley102（带青色荧光蛋白,CFP）和pEarley101（带黄色荧光蛋白,YFP）同源重组.将构建好的重组载体分别转化到农杆菌EHA105中,通过农杆菌接种法将两者共同注射到本氏烟叶片表皮细胞中进行表达.在共聚焦显微镜下观察,NS2-CFP与Atcoilin-YFP能重叠在一起,表明RSV NS2定位于柯浩体.     

单粒微米珠-单根DNA复合物的微流控分离初步研究

定点单分子DNA芯片有重要用途,但其制备极具挑战性,用光镊、磁镊及原子力显微镜等方法分离单分子DNA,为此建立的平台设备昂贵,且分离效率太低,难以满足实验要求。本研究采用微流控技术,在层流基础上电泳分离与富集单粒微米珠-单根DNA的复合物,旨在为基于微米孔阵列承载这种复合物的定点单分子DNA芯片的制备提供样本。此技术的完善将为单分子DNA芯片制作开辟新的途径。     

Reproducible measurement of single-molecule conductivity

A reliable method has been developed for making through-bond electrical contacts to molecules. Current-voltage curves are quantized as integer multiples of one fundamental curve, an observation used to identify single-molecule contacts. The resistance of a single octanedithiol molecule was 900 +/- 50 megohms, based on measurements on more than 1000 single molecules. In contrast, nonbonded contacts to octanethiol monolayers were at least four orders of magnitude more resistive, less reproducible, and had a different voltage dependence, demonstrating that the measurement of intrinsic molecular properties requires chemically bonded contacts.     

Room-temperature detection of a single molecule's absorption by photothermal contrast

So far, single-molecule imaging has predominantly relied on fluorescence detection. We imaged single nonfluorescent azo dye molecules in room-temperature glycerol by the refractive effect of the heat that they release in their environment upon intense illumination. This photothermal technique provides contrast for the absorbing objects only, irrespective of scattering by defects or roughness, with a signal-to-noise ratio of ~10 for a single molecule in an integration time of 300 milliseconds. In the absence of oxygen, virtually no bleaching event was observed, even after more than 10 minutes of illumination. In a solution saturated with oxygen, the average bleaching time was of the order of 1 minute. No blinking was observed in the absorption signal. On the basis of bleaching steps, we obtained an average absorption cross section of 4 angstroms(2) for a single chromophore.     

Inducing and viewing the rotational motion of a single molecule

Tunneling electrons from the tip of a scanning tunneling microscope were used to induce and monitor the reversible rotation of single molecules of molecular oxygen among three equivalent orientations on the platinum(111) surface. Detailed studies of the rotation rates indicate a crossover from a single-electron process to a multielectron process below a threshold tunneling voltage. Values for the energy barrier to rotation and the vibrational relaxation rate of the molecule were obtained by comparing the experimental data with a theoretical model. The ability to induce the controlled motion of single molecules enhances our understanding of basic chemical processes on surfaces and may lead to useful single-molecule devices.     

Single-molecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder

We used a multiplexed approach based on flow-stretched DNA to monitor the enzymatic digestion of lambda-phage DNA by individual bacteriophage lambda exonuclease molecules. Statistical analyses of multiple single-molecule trajectories observed simultaneously reveal that the catalytic rate is dependent on the local base content of the substrate DNA. By relating single-molecule kinetics to the free energies of hydrogen bonding and base stacking, we establish that the melting of a base from the DNA is the rate-limiting step in the catalytic cycle. The catalytic rate also exhibits large fluctuations independent of the sequence, which we attribute to conformational changes of the enzyme-DNA complex.     

Extreme bendability of DNA less than 100 base pairs long revealed by single-molecule cyclization

The classical view of DNA posits that DNA must be stiff below the persistence length [<150 base pairs (bp)], but recent studies addressing this have yielded contradictory results. We developed a fluorescence-based, protein-free assay for studying the cyclization of single DNA molecules in real time. The assay samples the equilibrium population of a sharply bent, transient species that is entirely suppressed in single-molecule mechanical measurements and is biologically more relevant than the annealed species sampled in the traditional ligase-based assay. The looping rate has a weak length dependence between 67 and 106 bp that cannot be described by the worm-like chain model. Many biologically important protein-DNA interactions that involve looping and bending of DNA below 100 bp likely use this intrinsic bendability of DNA.     

Counting cytokinesis proteins globally and locally in fission yeast

We used fluorescence microscopy to measure global and local concentrations of 28 cytoskeletal and signaling proteins fused to yellow fluorescent protein (YFP) in the fission yeast Schizosaccharomyces pombe. Native promoters controlled the expression of these functional YFP fusion proteins. Fluorescence measured by microscopy or flow cytometry was directly proportional to protein concentration measured by quantitative immunoblotting. Global cytoplasmic concentrations ranged from 0.04 (formin Cdc12p) to 63 micromolar (actin). Proteins concentrated up to 100 times in contractile rings and 7500 times in spindle pole bodies at certain times in the cell cycle. This approach can be used to measure the global and local concentrations of any fusion protein.