Turn of Promotor DNA by cAMP Receptor Protein Characterized by Bead Model Simulation of Rotational Diffusion

Abstract

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (< 100 bp). The final values reflect the solvated structure with the same ‘solvation layer’ added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix- turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 Å. According to these data the overall bending angle of our longest DNA fragment is approximately 180°, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.     

Effect of Bending Strain on the Torsion Elastic Constant of DNA

The torsion constants of both circular and linear forms of the same 181 bp DNA were investigated by time-resolved fluorescence polarization anisotropy (FPA) of intercalated ethidium. The ratio of intrinsic ethidium binding constants of the circular and linear species was determined from the relative fluorescence intensities of intercalated and non-intercalated dye in each case. Possible changes in secondary structure were also probed by circular dichroism (CD) spectroscopy. Upon circularization, the torsion constant increased by a factor of 1.42, the intrinsic binding constant for ethidium increased by about fourfold, and the CD spectrum underwent a significant change. These effects are attributed to an altered secondary structure induced by the bending strain. Quantitative agreement between torsion constants obtained from the present FPA studies and previous topoisomer distribution measurements on circular DNAs containing 205 to 217 bp removes a long-standing apparent discrepancy between those two methods. After storage at 4°C for eight months, the torsion constant of the circular DNA increased by about 1.25-fold, whereas that of the linear DNA remained unchanged. For these aged circles, both the torsion constant and intrinsic binding constant ratio lie close to the corresponding values obtained previously for a 247 bp DNA by analyzing topoisomer distributions created in the presence of various amounts of ethidium. The available evidence strongly implies that torsion constants measured for small circular DNAs with less than 250 bp are specific to the altered secondary structure(s) therein, and are not applicable to linear and much larger circular DNAs with lower mean bending strains.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Determination of the extent of DNA bending by an adenine-thymine tract

We determined the magnitude of the bend induced in DNA by an adenine-thymine tract by measuring the rate of cyclization of DNA oligonucleotides containing phased A tracts. A series of linear multimers with 2-bp single-stranded ends, in which the (A.T)6 tracts are separated by CG2-3C sequences and are positioned 10 and 11 bp apart alternately, were prepared from 21 bp long synthetic duplexed deoxyoligonucleotides. The cyclization rates of the multimers (105-210 bp) and the bimolecular association rate of the 84 bp long multimer were measured in the presence of DNA ligase. From the rate constants of the cyclization and bimolecular association reactions, ring closure probabilities were obtained for the multimers. The systematically bent molecules were simulated by Monte Carlo methods, and the ring closure probabilities were calculated for a given set of junction bend angles. By comparing the calculated values of ring closure probabilities to experimental values and adjusting the junction bend angles to fit experimental values, the extent of bending at the junctions (or the extent of bending for an adenine tract) was determined. We conclude that an A6 tract bends the DNA helix by 17-21 degrees.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

NF-κB决定小鼠胎肝激酶-1基因启动子的基本活性

为克隆小鼠胎肝激酶-1（fetal liver kinase-1,FLK-1）基因上游启动子序列,并观察其不同截短片段在小鼠血管内皮细胞中的启动子活性,以小鼠全基因组为模板,通过PCR扩增方法获得-258～+299 bp、-96～+299 bp、-71～+299 bp、-36～+299 bp大小的FLK-1启动子片段,将其定向克隆入pGL3 Basic,构建荧光素酶报告基因载体,并制备NF -κB结合位点的突变或缺失体.在阳离子脂质体介导下,报告基因载体瞬时转染小鼠血管内皮细胞株SVEC 4-10.结果发现,在小鼠血管内皮细胞中,各FLK-1启动子片段均有活性;－71～－36 bp区存在FLK-1启动子的核心调控元件.针对该区域NF-κB结合位点进行突变或缺失,能导致启动子活性显著降低;凝胶电泳迁移率实验表明该区段能结合转录因子NF-κB.结果提示,成功克隆了在血管内皮细胞中具有活性的FLK-1上游启动子序列,NF-κB是决定其基本活性的重要转录因子,为进一步研究FLK-1基因的转录调控机制奠定了基础.     

Genomic organization of the canrep repetitive DNA in Brassica juncea

Canrep is a heterogeneous, tandemly repeated, 176 bp nucleotide sequence that contains a single Hind III site and is present in high copy numbers in the genomes of many Brassica species. Complete clusters of repeats of this DNA were cloned from the nuclear DNA of Brassica juncea. Restriction-fragment dimers and higher multimers of the 176 bp sequence have arisen by mutations within the Hind III recognition sequence. Adjacent repeats from within the same cluster usually have different nucleotide sequences with features indicating that diversity is generated by a mechanism that causes site-specific base substitutions. While most of the units of canrep DNA are clustered in long arrays of tandem repeats, some are dispersed throughout the genome as isolated copies or in small clusters. Regardless of the size of the arrays, each cluster begins and ends with a variable-length, truncated repeat and is flanked by inverted copies of the sequence 5-ATCTCAT3-,which is not part of the basic sequence of the canrep family of DNAs. Furthermore, some clusters are located close to nucleotide sequences related to those of known plant transposons. Thus, canrep elements may be dispersed by transposition. There are two distinct subfamilies of canrep sequences in B. juncea, and one of these is closely related to one of the two subfamilies of this type of DNA from B. napus, indicating that it originated from B. campestris, the common diploid ancestor of both amphidiploid species. Neither the repetitive DNA nor nucleotide sequences flanking canrep clusters are transcribed in seedlings, suggesting that even small arrays of repeats are located in heterochromatic regions and might be involved in chromatin condensation and/or chromosome segregation.     

Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not further cut during uptake, the endonucleolytic activity associated with entry of plasmid DNA appeared to act preferentially on circular DNA. Although linear plasmid DNA was taken up into a DNase-resistant state as efficiently as circular DNA, linear plasmid DNA transformed much less efficiently than circular plasmid DNA. These data suggest that during entry transforming plasmid DNA often is processed to double-stranded linear molecules; transformants may arise when some molecules are repaired to form circles. Occasional molecules which enter as intact circles may also lead to transformants.     

<Emphasis Type="Italic">PpVIN2</Emphasis>, an acid invertase gene family member,is sensitive to chilling temperature and affects sucrose metabolism in postharvest peach fruit

We previously reported that expression and activity of acid invertases (AI) are increased in peach fruit under chilling stress. In order to determine which AI genes respond to chilling stress, seven AI genes, two vacuolar invertases (VINs) and five cell wall-bound invertases (CWINs), were identified and cloned. The predicted amino acid sequences of the genes contain conserved sites characteristic of plant AIs such as NDPNG/A, the sucrose-binding site, and MWECV/P, a cysteine catalytic motif. Using gene-specific primers, the expression of each gene was measured in ‘Baifeng’ and ‘Yulu’ peach fruits stored at 0, 5, 10 and 20 °C. Of the seven genes, expression of PpVIN2 was the most affected by chilling stress; the largest increases were in fruit stored at 5 °C, up to 17-fold in ‘Baifeng’ fruit, and up to 280-fold in ‘Yulu’ fruit. Overall, VIN activity was much higher than CWIN activity in stored peach fruit. In both cultivars reducing sugar content increased significantly and sucrose content decreased gradually during storage at 5 °C relative to other temperatures, and was accompanied by severe chilling injury symptoms. Thus, PpVIN2 appears to be induced by chilling and may play an important role in sucrose metabolism in peach fruit subjected to cold storage.     

Extracting DNA from museum bird eggs, and whole genome amplification of archive DNA

We present a comprehensive protocol for extracting DNA from egg membranes and other internal debris recovered from the interior of blown museum bird eggs. A variety of commercially available DNA extraction methods were found to be applicable. DNA sequencing of polymerase chain reaction (PCR) products for a 176‐bp fragment of mitochondrial DNA was successful for most egg samples (> 78%) even though the amount of DNA extracted (mean = 14.71 ± 4.55 ng/µL) was significantly less than that obtained for bird skin samples (mean = 67.88 ± 4.77 ng/µL). For PCR and sequencing of snipe (Gallinago) DNA, we provide eight new primers for the ‘DNA barcode’ region of COI mtDNA. In various combinations, the primers target a range of PCR products sized from 72 bp to the full ‘barcode’ of 751 bp. Not all possible combinations were tested with archive snipe DNA, but we found a significantly better success rate of PCR amplification for a shorter 176‐bp target compared with a larger 288‐bp fragment (67% vs. 39%). Finally, we explored the feasibility of whole genome amplification (WGA) for extending the use of archive DNA in PCR and sequencing applications. Of two WGA approaches, a PCR‐based method was found to be able to amplify whole genomic DNA from archive skins and eggs from museum bird collections. After WGA, significantly more archive egg samples produced visible PCR products on agarose (56.9% before WGA vs. 79.0% after WGA). However, overall sequencing success did not improve significantly (78.8% compared with 83.0%).     

人类RELM-beta基因启动子的结构与功能分析

为克隆人类抵抗素样分子β（resistin-like molecule beta, RELMβ）基因的上游启动子序列,并观察其不同截短片段的启动子活性,以人类基因组DNA为模板,通过PCR扩增方法获得-871～+50 bp、-729～+50 bp、-471～+50 bp、-438～+50 bp、-371～+50 bp大小的RELMβ启动子片段,将其定向克隆入pGL3-Basic载体,构建荧光素酶报告基因载体,并制备转录因子CDX-2结合位点的突变或缺失体.在阳离子脂质体的介导下,报告基因载体分别瞬时转染人胚肾293细胞、结肠癌HCT116和SW480细胞、宫颈癌HeLa细胞.结果发现,各RELMβ启动子片段在293、HCT116、SW480细胞中均有活性,但在HeLa细胞中活性缺失;-471～-438 bp区存在RELMβ启动子的核心调控元件.针对该区域CDX-2转录因子结合位点进行突变,能导致RELMβ启动子活性显著降低;凝胶电泳迁移率实验表明,该区段能结合CDX-2.结果提示,成功克隆了具有活性的RELMβ启动子序列,CDX-2为其重要的转录因子,为研究RELMβ基因的转录调控机制奠定了实验基础.     

Analysis of Junction Sequences Resulting from Integration at Nonhomologous Loci in Neurospora Crassa

We have analyzed the junctions involved in two examples of ectopic integration of plasmids containing the am+ (glutamate dehydrogenase) gene into a strain of Neurospora crassa bearing a complete deletion of the am locus. In one transformed strain a single copy of plasmid DNA had been integrated into linkage group (LG) III DNA without the loss of chromosomal DNA. In contrast, 450 bp had been lost from plasmid sequences at the site of integration. The transforming DNA used was circular, so we postulate that the plasmid was linearized and truncated prior to its integration by end joining into a break in LG III DNA. There was no significant homology between the incoming DNA and DNA at the site of integration. The second transformed strain resulted from transformation with a linearized plasmid. It contained multiple integrated copies of plasmid DNA, one of which was recloned, together with adjacent chromosomal DNA, by plasmid rescue in Escherichia coli. Prior to integration into chromosomal DNA, the linear plasmid had been truncated by 64 bp on one end and 3.2 kbp on the other end. One end of the integrated DNA was adjacent to DNA from the right arm of LG I, while the other end was integrated into a copy of a repetitive sequence. Restriction fragment length polymerism mapping showed that integration was in a copy of the repetitive sequence that is linked to the previously unassigned telomere M11 and is distantly linked to the LG VI marker con-11. Genetic analysis revealed that a long segment of LG I containing all markers from un-1 to the right tip has been translocated to the right end of LG VI. Tetrad analysis showed that the integrated DNA was closely linked to the translocation. We conclude that the transforming DNA was truncated and joined to DNA from two different chromosomes by end joining during the formation of a quasiterminal translocation, T(IR----VIR) UK-T12. We also conclude that the previously unassigned telomere, M11, is the right end of LG VI.     

DNA gyrase can supercoil DNA circles as small as 174 base pairs.

DNA gyrase introduces negative supercoils into closed-circular DNA using the free energy of ATP hydrolysis. Consideration of steric and thermodynamic aspects of the supercoiling reaction indicates that there should be a lower limit to the size of DNA circle which can be supercoiled by gyrase. We have investigated the supercoiling reaction of circles from 116-427 base pairs (bp) in size and have determined that gyrase can supercoil certain relaxed isomers of circles as small as 174 bp, dependent on the final superhelix density of the supercoiled product. Furthermore, this limiting superhelical density (-0.11) is the same as that determined for the supercoiling of plasmid pBR322. We also find that although circles in the range 116-152 bp cannot be supercoiled, they can nevertheless be relaxed by gyrase when positively supercoiled. These data suggest that the conformational changes associated with the supercoiling reaction can be carried out by gyrase in a circle as small as 116 bp. We discuss these results with respect to the thermodynamics of DNA supercoiling and steric aspects of the gyrase mechanism.     

Formation of small circular DNA molecules via an in vitro site-specific recombination system

The Cre-lox site-specific recombination system of bacteriophage P1 has been used to investigate the role of DNA flexibility in recombination. We have determined that a minimal distance of 82 bp must separate two loxP sites located on the same DNA molecule to allow these sites to undergo intramolecular recombination with one another. As a result of recombination, DNA circles as small as 116bp have been produced. In addition, we have demonstrated that the nuclease BAL 31 recognizes distortions in the DNA helix resulting from the formation of small DNA circles whose length is not a multiple of the helical repeat.     

Stereoselective Covalent Binding of Anti-benzo(a)pyrene Diol Epoxide to DNA Conformation of Enantiomer Adducts

Abstract

The conformation of adducts derived from the reactions and covalent binding of the (+) and (-) enantiomers of 7β, 8α-dihydroxy-9α, 10α-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (anti-BaPDE) with double-stranded calf thymus DNA in vitro were investigated utilizing the electric linear dichroism technique. The linear dichroism and absorption spectra of the covalent DNA complexes are interpreted in terms of a superposition of two types of binding sites. One of these conformations (site I) is a complex in which the plane of the pyrene residue is close to parallel (within 30°) to the planes of the DNA bases (quasi-intercalation), while the other (site II) is an external binding site; this latter type of adduct is attributed to the covalent binding of anti-BaPDE to the exocyclic amino group of deoxyguanine (N2-dG), while site I adducts are attributed to the 06-deoxyguanine and N6-deoxyadenine adducts identified in the product analysis of P. Brookes and M.R. Osborne (Carcinogenesis (1982) 3, 1223–1226). Site II adducts are dominant (~90% in the covalent complexes derived from the (+) enantiomer), but account for only 50±5% of the adducts in the case of the (—)-enantiomer. The orientation of site II complexes is different by 20±10° in the adducts derived from the binding of the (+) and the (—) enantiomers to DNA, the long axis of the pyrene chromophore being oriented more parallel to the axis of the DNA helix in the case of the (+) enantiomer. These findings support the proposals by Brookes and Osborne that the difference in spatial orientation of the N2-dG adducts of (-)-anti-BaPDE together with their lower abundance may account for the lower biological activity of the (—) enantiomer. The external site II adducts, rather than site I adducts, appear to be correlated with the biological activity of these comoounds.     

The freshwater littoral meiofauna in a South Carolina reservoir receiving thermal effluents*

SUMMARY. The freshwater littoral meiofauna along a temperature gradient in Par Pond (a cooling reservoir receiving thermal effluents from a nuclear reactor) was sampled from September 1975 to October 1976. Monthly samples were taken at three stations; ‘hot-water’ (15–40°C), ‘warm-water’ (13–37°C) and ambient or ‘cold-water’ (8–37°C) sites. Total numbers of individuals at the ambient site ranged from 971 to 3674 per 10 cm² (mean = 2263), approximating the density reported from productive estuarine environments. Nematodes, rotifers, ostracods, cladocerans and mites comprised 80% of overall density. When compared to the ambient site, thermally affected sites demonstrated reduced faunal density. Contrary to other environmental perturbation studies, the Shannon-Weaver diversity index (H') did not reflect alteration of structural complexity within the rotifer taxocene when hot-water, warm-water and cold-water sites were compared. Though there was a significant reduction in number of species at the thermally altered sites, high ‘equitability’ among the reduced species resulted in H’ values comparable to those in natural communities. Distinct winter-spring and summer-autumn assemblages were evident in the ambient community while no seasonal clustering was apparent at the thermally affected sites. It is hypothesized that the maintenance of temperatures above ambient normals masks environmental cues that normally would elicit seasonal changes in the rotifer fauna at those two sites.     

Separation of Different Conformations of Plant Mitochondrial DNA Molecules by Field Inversion Gel Electrophoresis

Mitochondrial (mt) DNA structure in higher plants is still unclear as to the circularity or linearity of the genome. We have developed a system to electrophoretically separate distinct populations of mtDNA, with some populations enriched for networked linear and circular DNA molecules. Using field inversion gel electrophoresis (FIGE) and electron microscopy (EM), we have identified four distinct populations of mtDNA from two Brassica species. Using FIGE, two slow migrating mtDNA populations ran faster than a 66 kbp Escherichia coli circular plasmid marker, while these same populations comigrated in the compression zone in contour-clamped homogeneous electrophoretic field (CHEF) gels. A fast-migrating mtDNA population was also resolved by FIGE as a diffuse band between 20 to 70 kbp when compared with linear lambda () markers. FIGE resolved the 66 kbp circular marker into several multimers, while CHEF resolved only open-circular monomers and linears. In agreement with FIGE results, EM analysis indicated the two slow migrating mtDNA populations contained circular (both supercoiled and relaxed circles) and free linear molecules of 10-60 kbp, and networked linear molecules of 45–140 kbp total size that may represent recombination intermediates. The fast migrating population consisted of 10–50 kbp linear molecules. Well-bound mtDNA showed only long linear molecules of 40–150 kbp with no detection of circles or complex/rosette molecules. This report shows that FIGE has clear advantages over CHEF for separating large DNA molecules with different conformations, and may be very useful for studies to characterize genome structure in complex systems such as plant mitochondria.     

Three-channel Lissajous′ trajectories of auditory event-related potentials to target stimuli

Three orthogonal recordings (‘X’, ‘Y’ and ‘Z’) of event-related potentials (ERPs) evoked by auditory target stimuli were represented in 3-dimensional voltage-space, to produce 3-channel Lissajous′ trajectories (3-CLTs). Stimuli were verbal or non-verbal and were differentiated by their pitch, phonemic or phonetic attributes. Visual inspection and quantitative evaluations unexpectedly revealed that the 3-CLT of these ERPs strongly resembles a ‘hair-pin’ trajectory. This trajectory tilted in space at characteristic angles with each of the analysis axes: 133° with the ‘X’ axis, 87° with the ‘Y’ axis, and 54° with the ‘Z’ axis. The relatively small inter-subject variability observed in the geometrical measures, particularly in orientation, may be attributed to the slight variation in the underlying generators. 3-CLT analysis could be useful in future clinical as well as source studies of ERP generators.