The hierarchical approach to the DNA stability problem. II. Some applications and speculations with yeast mitochondrial DNA as an example

As discussed in the preceding article [1] hierarchical analysis of DNA sequences should make it possible to treat complex unfolding (and refolding) processes involving both equilibrium and non-equilibrium subtransitions. Hence a variety of actual experimental situations may be analyzed. This is demonstrated with the help of a 1950 bp yeast mitochondrial DNA sequence encompassing part of the 21S ribosomal RNA gene: excellent fit of complex denaturation and renaturation profiles is achieved with only two adjustable parameters. The advantage of dealing with objectively defined stability units is also apparent when stability profiles are compared to known functional maps: striking correlations may be brought out and their possible significance is briefly discussed.     

A method for measuring microquantities of DNA

Quantitative analysis of DNA content represents a critical step when only very small amounts of nucleic acids are available. The DNA content of a small RNA-free sample can be measured in a simple and precise way using a two-dimensional approach. DNA samples are spotted on the surface of an agarose gel containing ethidium bromide (EtBr) and the ultraviolet-induced low-light fluorescence emitted by EtBr molecules intercalated into the DNA is evaluated. The high sensitivity and reproducibility of this quantitative method has been obtained using an advanced analysis system capable of distinguishing low-light fluorescent patterns, as in the case of DNA stained with EtBr, from the background. Use of an internal standard is necessary because the intensity of the signal is due to the aperture of camera diaphragm and to gel conditions. Using this two-dimensional analysis system it is possible to obtain rapid and precise quantitation of as little as 2ng of DNA.     

MICROFLUOROMETRIC MEASUREMENT OF DNA IN EUDORINA ELEGANS AND E. CALIFORNICA (CHOROPHYCEAE)1

The fluorescent dye 2,5-bis (4′-aminophenyl (1′))-1,3,4-oxidiwle (BAO) was compared to the Feulgen procedure as a method for quantitative comparison of DNA levels in two species of Eudorina. The BAO procedure proved to be specific and as quantitative as the Feulgen procedure, and was more convenient and rapid. The DNA level of the nuclei of gonidial cells of Eudorina elegans Ehrenberg and E. californica (Shaw) Goldstein doubled prior to each division. The DNA level of the somatic, usually non-dividing nuclei of E. californica did not vary from the IC (haploid) level.     

Synchrotron excitation of DNA fluorescence. Decay time evidence for excimer emission at room temperature

The first lifetime measurements of DNA fluorescence are reported. Natural and synthetic DNA have been excited by 1.76 ns pulses of synchrotron ultraviolet radiation (270 nm) and the time profile of the fluorescence has been measured by synchronous single-photon counting. A post-pulse exponentially decaying emission has been observed with a lifetime of 2.9 +/- 0.4 ns for calf thymus DNA and 3.0 +/- 0.3 ns for poly(dA-T); this is most likely an excimer fluorescence.     

Time resolved fluorescence of bacteriophage Pfl DNA binding protein and its complex with DNA

The DNA binding protein of the filamentous bacteriophage Pfl exhibits fluorescence from a single tryptophan residue. The location of the emission maximum at 340 nm ist quite common for proteins, but the single lifetime of 7.8 ns is one of the longest yet reported. Protein fluorescence is quenched more efficiently by Cs⁺ than by I^-; the Trp is located in a partially exposed pocket, in the vicinity of a negative charge.In the native complex of the binding protein with Pfl DNA the fluorescence emission maximum is at 330 nm, indicating a more apolar environment for Trp 14. The native nucleoprotein complex exhibits a similar fluorescence lifetime (6.5 ns) and an approximately equal fluorescence yield, indicating the absence of Trp-DNA stacking. The tryptophan in the complex is virtually inaccessible to ionic quenchers, and thus appears to be buried.Fluorescence depolarisation measurements have been used to examine the rotational mobility of the tryptophan in the protein and in the nucleoprotein complex. In the protein alone a single rotational correlation time () of 19 ns is observed, corresponding to rotation of the entire dimeric molecule; in the native nucleoprotein complex with Pfl DNA, a of 500 ns is observed, corresponding to a rigid unit of at least 50 subunits. In neither case does the tryptophan exhibit any detectable flexibility on the subnanosecond time scale.     

基于流式细胞技术的灵芝基因组大小估测

以药典规定的灵芝正品来源灵芝Ganoderma lucidum作为研究对象,利用已完成全基因组测序的黑曲霉Aspergillus niger作为内标,通过机械破碎菌丝体的方法获得合适浓度的细胞核悬液,碘化丙啶荧光染色后成功应用流式细胞术进行基因组大小估测。经过优化材料培养、样品制备、上机分析等实验条件,估测得出灵芝基因组大小(48.98±0.60)Mb,为灵芝基因组学研究提供重要数据。该方法简捷稳定,在蕈菌范围内,首次得到了全基因组测序数据与光学图谱结果验证,为蕈菌基因组学研究提供重要技术平台。     

DNA bioassays based on the fluorescence ‘turn off’ of silver nanocluster beacon

Recognition and quantification of oligonucleotide sequences play important roles in medical diagnosis. In this study, a new fluorescent oligonucleotide‐stabilized silver nanocluster beacon (NCB) probe was designed for sensitive detection of oligonucleotide sequence targets. This probe contained two tailored DNA strands. One strand was a signal probe strand containing a cytosine‐rich strand template for fluorescent silver nanocluster (Ag NC) synthesis and a detection sections at each end. The other strand was a fluorescence enhancing strand containing a guanine‐rich section for signal enhancement at one end and a linker section complementary to one end of the signal probe strand. After synthesis of the Ag NCs and hybridization of the two strands, the fluorescence intensity of the as‐prepared silver NCB was enhanced 200‐fold compared with the Ag NCs. Two NCBs were designed to detect two disease‐related oligonucleotide sequences, and results indicated that the two target oligonucleotide sequences in the range 50.0–600.0 and 50.0–200.0 nM could be linearly detected with detection limits of 20 and 25 nM, respectively. The developed fluorescence method using NCBs for oligonucleotide sequence detection was sensitive, facile and had potential for use in bioanalysis and diagnosis.     

荧光偏振技术研究Bloom解旋酶催化核心与双链DNA的相互作用

Bloom 综合症(BLM)解旋酶是RecQ家族DNA解旋酶中的一个重要成员,参与了DNA复制、修复、转录、重组以及端粒的维持等细胞代谢过程,在维持染色体的稳定性中具有重要的作用．BLM解旋酶的突变可导致Bloom综合症,患者遗传不稳定易患多种类型癌症．本研究运用荧光偏振技术研究BLM解旋酶催化核心(BLM^642～1290)与双链DNA(dsDNA)的相互作用,分析其相关特征参数,了解BLM^642～1290解旋酶与dsDNA的结合和解链特性．结果表明：BLM^642～1290解旋酶与dsDNA的结合和解链与dsDNA 3′端的单链DNA(ssDNA)长度有关;解旋酶优先结合于dsDNA底物的ssDNA末端,且每分子解旋酶可结合9.6 nt的ssDNA;dsDNA 3′端ssDNA的长度为9.6 nt时,解旋酶的解链效率达到最大且不再随其长度而变化．另外,BLM^642～1290解旋酶也能够结合和解链钝末端dsDNA,但其结合亲和力和解链效率低于有3′端ssDNA的dsDNA．推测BLM^642～1290解旋酶在与dsDNA底物结合和解链时是单体形式,可能以尺蠖的形式解开dsDNA．这些结果可为进一步研究BLM解旋酶的功能特征提供理论基础．     

Ligands differentially modulate the protein interactions of the human estrogen receptors alpha and beta

The interactions of human estrogen receptor subtypes ERalpha and ERbeta with DNA and a 210 amino acid residue fragment of the coactivator protein SRC-1 bearing three nuclear receptor interaction motifs were investigated quantitatively using fluorescence anisotropy in the presence of agonist and antagonist ligands. ERalpha and ERbeta were found to bind in a similar manner to DNA, and both salt and temperature affected the affinity and/or stoichiometry of these interactions. The agonist ligands estradiol, estrone and estriol did not modify the binding of ERalpha to the fluorescein-labeled target estrogen response element. However, in the case of ERbeta, these ligands led to the formation of some higher-order protein-DNA complexes and a small decrease in affinity. The partial agonist 4-hydroxytamoxifen had little effect on either ER subtype, whereas the pure antagonist ICI 182,780 led to the cooperative formation of protein-DNA complexes of higher order than dimer, as further demonstrated by competition experiments and gel mobility-shift assays. In addition to DNA binding, the interaction of both ER subtypes with the Alexa488-labeled SRC-1 coactivator fragment was investigated by fluorescence anisotropy. The agonist ligands estrone, estradiol, estriol, genistein and ethynyl estradiol exhibited distinct capacities for inducing the recruitment of SRC-1 that were not correlated with their affinity for the receptor. Moreover, estrone and genistein exhibited subtype specificity in that they induced SRC-1 recruitment to ERbeta with much higher efficiency than in the case of ERalpha. The differential coactivator recruitment capacities of the ER agonists and their receptor subtype coactivator recruitment specificity may be linked to the molecular structure of the agonists with respect to their interactions with a specific histidine residue located at the back of the ligand-binding pocket. Altogether, these quantitative in vitro studies of ER interactions reveal the complex energetic and stoichiometric consequences of changes in the chemical structures of these proteins and their ligands.     

DNA hybridization assays using metal-enhanced fluorescence

We describe a new approach to DNA hybridization assays using metal-enhanced fluorescence. Thiolated oligonucleotides were bound to silver particles on a glass substrate. Addition of a complementary fluorescein-labeled oligonucleotide resulted in a dramatic time-dependent 12-fold increase in fluorescence intensity during hybridization. Proximity to silver particles resulted in a decreased fluorescence lifetime. This effect is thought to be the result of enhanced fluorescence from fluorescein near metallic silver particles. Hybridization could thus be measured from the decay kinetics of the emission, which can be measured independently from the emission intensity. These results suggest the use of silver particles as a general approach to measure DNA hybridization as a method to increase the sensitivity of DNA detection.     

Unmethylated and methylated CpG dinucleotides distinctively regulate the physical properties of DNA

In eukaryotic cells, DNA has to bend significantly to pack inside the nucleus. Physical properties of DNA such as bending flexibility and curvature are expected to affect DNA packaging and partially determine the nucleosome positioning patterns inside a cell. DNA CpG methylation, the most common epigenetic modification found in DNA, is known to affect the physical properties of DNA. However, its detailed role in nucleosome formation is less well‐established. In this study, we evaluated the effect of defined CpG patterns (unmethylated and methylated) on DNA structure and their respective nucleosome‐forming ability. Our results suggest that the addition of CpG dinucleotides, either as a (CG)_n stretch or (CGX₈)_n repeats at 10 bp intervals, lead to reduced hydrodynamic radius and decreased nucleosome‐forming ability of DNA. This effect is more predominant for a DNA stretch ((CG)₅) located in the middle of a DNA fragment. Methylation of CpG sites, surprisingly, seems to reduce the difference in DNA structure and nucleosome‐forming ability among DNA constructs with different CpG patterns. Our results suggest that unmethylated and methylated CpG patterns can play very different roles in regulating the physical properties of DNA. CpG methylation seems to reduce the DNA conformational variations affiliated with defined CpG patterns. Our results can have significant bearings in understanding the nucleosome positioning pattern in living organisms modulated by DNA sequences and epigenetic features. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 517–524, 2014.     

Long,Processive Enzymatic DNA Synthesis Using 100% Dye-Labeled Terminal Phosphate-Linked Nucleotides

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by φ29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.     

Nucleotides sequestered at different subsite loci within DNA‐binding pockets of two OB‐fold single‐stranded DNA‐binding proteins are unstacked to different extents

The gene 5 protein (g5p) encoded by the Ff strains of Escherichia coli bacteriophages is a dimeric single‐stranded DNA‐binding protein (SSB) that consists of two identical OB‐fold (oligonucleotide/oligosaccharide‐binding) motifs. Ultrafast time‐resolved fluorescence measurements were carried out to investigate the effect of g5p binding on the conformation of 2‐aminopurine (2AP) labels positioned between adenines or cytosines in the 16‐nucleotide antiparallel tails of DNA hairpins. The measurements revealed significant changes in the conformational heterogeneity of the 2AP labels caused by g5p binding. The extent of the changes was dependent on sub‐binding‐site location, but generally resulted in base unstacking. When bound by g5p, the unstacked 2AP population increased from ～22% to 59–67% in C‐2AP‐C segments and from 39% to 77% in an A‐2AP‐A segment. The OB‐fold RPA70A domain of the human replication protein A also caused a significant amount of base unstacking at various locations within the DNA binding site as evidenced by steady‐state fluorescence titration measurements using 2AP‐labeled 5‐mer DNAs. These solution studies support the concept that base unstacking at most of a protein's multiple sub‐binding‐site loci may be a feature that allows non‐sequence specific OB‐fold proteins to bind to single‐stranded DNAs (ssDNAs) with minimal preference for particular sequences. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 484–496, 2013.     

Fluorescence enhancement of fluorophores tethered to different sized silver colloids deposited on glass substrate

We studied fluorescence enhancements of fluorescein tethered to silver colloids of different size. Thiolated 23-mer oligonucleotide (ss DNA-SH) was bound selectively to silver colloids deposited on 3-aminopropyltriethoxysilane (APS)-treated quartz slides. Fluorescein-labeled complementary oligonucleotide (ss Fl-DNA) was added in an amount significantly lower than the amount of unlabeled DNA tethered to the colloids. The hybridization kinetics, observed as an increase in fluorescence emission, on small (30-40 nm) and large (> 120 nm) colloids were similar. However, the final fluorescence intensity of the sample with large colloids was about 50% higher than that observed for the sample with small colloids. The reference sample without ss DNA-SH was used to estimate the fluorescence enhancements of fluorescein tethered to the small colloids (E = 2.7) and to the large colloids (E = 4.1) due to its steady fluorescence signal. The proposed method, based on controlled hybridization with minimal amount of fluorophore labeled ss DNA, can be used to reliably estimate the fluorescence enhancements on any silver nanostructures.     

Rapid analysis of amplified double-stranded DNA by capillary electrophoresis with laser-induced fluorescence detection

DNA amplification technology has been applied to clinical diagnosis of infectious disease, genetic disorder, and cancer. After in vitro amplification of a particular DNA region, the methods of analysis for these amplified samples play a pivotal role in clinical diagnosis. Conventional gel electrophoresis has been routinely used in the lab for checking DNA. The whole procedure is time consuming and requires more than 1 ng of DNA for detection. To achieve greater performance in DNA diagnosis, we demonstrated capillary electrophoresis with laser induced fluorescence detection for analysis of amplified DNA. The analysis of DNA could be completed within 3 min and the data is directly entered into the computer. Considering the automatic and rapid process, we believe that this method could be routinely utilized for the clinical diagnosis of amplified DNA products.     

Binding of two PriA-PriB complexes to the primosome assembly site initiates primosome formation

A direct quantitative analysis of the initial steps in primosome assembly, involving PriA and PriB proteins and the minimal primosome assembly site (PAS) of phage ?X174, has been performed using fluorescence intensity, fluorescence anisotropy titration, and fluorescence resonance energy transfer techniques. We show that two PriA molecules bind to the PAS at both strong and weak binding sites on the DNA, respectively, without detectable cooperative interactions. Binding of the PriB dimer to the PriA-PAS complex dramatically increases PriA's affinity for the strong site, but only slightly affects its affinity for the weak site. Associations with the strong and weak sites are driven by apparent entropy changes, with binding to the strong site accompanied by a large unfavorable enthalpy change. The PriA-PriB complex, formed independently of the DNA, is able to directly recognize the PAS without the preceding the binding of PriA to the PAS. Thus, the high-affinity state of PriA for PAS is generated through PriA-PriB interactions. The effect of PriB is specific for PriA-PAS association, but not for PriA-double-stranded DNA or PriA-single-stranded DNA interactions. Only complexes containing two PriA molecules can generate a profound change in the PAS structure in the presence of ATP. The obtained results provide a quantitative framework for the elucidation of further steps in primosome assembly and for quantitative analyses of other molecular machines of cellular metabolism.     

Thermodynamic dissection of the polymerizing and editing modes of a DNA polymerase

DNA polymerases with intrinsic proofreading activity interact with DNA primer/templates in two distinct modes, corresponding to the complexes formed during the 5'-3' polymerization or 3'-5' editing of a nascent DNA chain. Thermodynamic measurements designed to quantify the energetic contributions of individual DNA-protein contacts in either the polymerizing or editing complexes are complicated by the fact that both species exist in solution and are not resolved in conventional DNA-protein binding assays. To overcome this problem, we have developed a new binding analysis that combines information from steady-state and time-resolved fluorescence experiments and uses the Klenow fragment of Escherichia coli DNA polymerase I (KF) and fluorescently labeled primer/template oligonucleotides as a model polymerase-DNA system. Steady-state fluorescence titrations are used to evaluate the overall affinity of KF for the primer/template, while time-resolved fluorescence anisotropy is used to quantify the equilibrium fractions of the primer/template bound in the polymerizing and editing modes. From a combined analysis of both data, the equilibrium constant and hence standard free energy change associated with each binding mode can be obtained unequivocally. This method is initially used to determine the equilibrium constants describing binding of a correctly base-paired primer/template to the 5'-3' polymerase and 3'-5' exonuclease sites of KF. It is then extended to quantify the extent to which these parameters are affected by the introduction of mismatches into the primer/template, and by rearrangement of specific side-chains in the exonuclease domain of the protein. While these perturbants were originally designed to demonstrate the utility of our new approach, they are also relevant in their own right since they have helped identify some hitherto unknown determinants of polymerase fidelity.     

一株能在苜蓿上结瘤的费氏中华根瘤菌

费氏中华根瘤菌 (Sinorhizobiumfredii) 0 4 2BS分离自新疆的苜蓿根瘤 ,通过交叉结瘤试验 ,发现它既可在苜蓿上又可在大豆上结瘤固氮。 1 6SrDNAPCR RFLP分析表明 ,0 4 2BS与费氏中华根瘤菌模式菌株USDA2 0 5的 4种限制性酶切图谱完全一致。其G +Cmol%为 60 0 ,与费氏中华根瘤菌USDA2 0 5和USDA1 91的DNA同源性分别为 84 9%和89 6% ,表明 0 4 2BS属于费氏中华根瘤菌。应用绿色荧光蛋白基因标记 0 4 2BS ,得到重组菌株 0 4 2BSG。将其接种保定苜蓿和北引 1号大豆 ,并重新分离出根瘤菌 ,利用激光共聚焦荧光显微镜检测到标记基因的表达 ,从而确证了 0 4 2BS能在苜蓿和大豆上结瘤。而且 ,0 4 2BS对不同苜蓿品种的结瘤能力不同