Structure of (dG)n · (dC)n Under Superhelical Stress and Acid pH

Abstract

We have recently shown that a (GA)_n · (TC)_n tract undergoes a sharp structural transition under superhelical stress (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 327 (1985)). Unlike the well studied transitions to the cruciform and to the Z form, this novel transition was strongly pH-dependent. We have found the (dG)_n · (dC)_n insert to undergo a pH-dependent structural transition similar to that of the (GA)_n · (TC)_n tract. These new data meet our earlier expectations and disagree with the data of D.E. Pulleyblank, D.B. Haniford and A.R. Morgan, Cell 42, 271 (1985). We conclude that a novel DNA structure (the H-form) is typical of homopurine-homopyrimidine mirror repeats (the H palindromes) under superhelical stress and/or acid pH. In the H-form the homopyrimidine strand forms a hairpin while half of the homopurine strand interacts with the hairpin forming a triplex, the other half of the homopurine strand being unstructured (V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 2, 3,667 (1986)).     

Sedimentation coefficient of polyoma virus DNA

The sedimentation coefficient of the twisted circular form of polyoma virus DNA is calculated from the Kirkwood sedimentation–diffusion equation, the structure being assumed to be a rigid double superhelix. Agreement with the experimental sedimentation coefficient can be obtained, with the use of an experimental value for the number of superhelical turns, when the pitch of the superhelix is intermediate between its minimal and maximal possible values. Another model, which has been proposed for polyoma DNA at low ionic strengths, may be visualized as a superhelical structure wound about a torus. Calculations of sedimentation coefficients for this model agree qualitatively with experimental data at ionic strengths Below 10^?2M.     

Structures of homopurine-homopyrimidine tract in superhelical DNA

For homopurine-homopyrimidine tracts in superhelical DNA, we propose a structure involving Watson-Crick and Hoogsteen paired triple helixes, hairpin loops and unstructured domains. Topologically, the whole structure is equivalent to an open region. The proposed structure is consistent with available S1 cleavage, pH and alkylation data and energetics under superhelical stress; this new structure is a much more probable candidate than the one proposed by us recently (V.I. Lyamichev, S.M. Mirkin & M.D. Frank-Kamenetskii, J. Biomole. Str. Dyns 3, 327-338, 1985).     

Unusual Protonated Structure in the Homopurine · Homopyrimidine Tract of Supercoiled and Linearized Plasmids Recognized by Chemical Probes

Abstract

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long · homopurine homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively.

At pH 6.0 the site-specific modification of the homopurine · homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine · homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine · homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3, 667 (1986).     

A pH-dependent Structural Transition in the Homopurine-homopyrimidine Tract in Superhelical DNA

Abstract

We have inserted the 509-bp-long fragment of sea urchin P. miliaris histone gene spacer region into plasmid pUC19. The fragment contains the 60-bp-long homopurine-homopyrimidine tract that is known to be hypersensitive to the S1 endonuclease. Using two-dimensional gel electrophoresis we have observed a sharp structural transition in the insert with increasing DNA superhelicity. As in the cases of cruciform and Z form formation, the observed transition partly relaxes the superhelical stress. In contrast with the other two well documented transitions, the observed transition strongly depends on pH. At pH7 and above the transition occurs at negative superhelicities exceeding the physiological range (σ>0.08). For pH6 the transition occurs at ?σ = 0.055, whereas for pH4.3 it takes place at ?σ = 0.001. A comprehensive analysis of the obtained data has made it possible to define the nature of the observed transition. We conclude that under superhelical stress or/and at low pH homopurine- homopyrimidine tracts adopt a novel spatial structure called the H form.     

Structural and Molecular Biology of the eye Lens Membrane

Abstract

The DNA double helix exhibits local sequence-dependent polymorphism at the level of the single base pair and dinucleotide step. Curvature of the DNA molecule occurs in DNA regions with a specific type of nucleotide sequence periodicities. Negative supercoiling induces in vitro local nucleotide sequence-dependent DNA structures such as cruciforms, left-handed DNA, multistranded structures, etc. Techniques based on chemical probes have been proposed that make it possible to study DNA local structures in cells. Recent results suggest that the local DNA structures observed in vitro exist in the cell, but their occurrence and structural details are dependent on the DNA superhelical density in the cell and can be related to some cellular processes.     

A relational theory of biological systems II

The general Theory of Categories is applied to the study of the (M, R)-systems previously defined. A set of axioms is provided which characterize “abstract (M, R)-systems”, defined in terms of the Theory of Categories. It is shown that the replication of the repair components of these systems may be accounted for in a natural way within this framework, thereby obviating the need for anad hoc postulation of a replication mechanism. A time-lag structure is introduced into these abstract (M, R)-systems. In order to apply this structure to a discussion of the “morphology” of these systems, it is necessary to make certain assumptions which relate the morphology to the time lags. By so doing, a system of abstract biology is in effect constructed. In particular, a formulation of a general Principle of Optimal Design is proposed for these systems. It is shown under what conditions the repair mechanism of the system will be localized into a spherical region, suggestive of the nuclear arrangements in cells. The possibility of placing an abstract (M, R)-system into optimal form in more than one way is then investigated, and a necessary and sufficient condition for this occurrence is obtained. Some further implications of the above assumptions are then discussed.     

Chromosomal supercoiling in Escherichia coli

The Escherichia coli chromosome is compacted into 40-50 negatively supercoiled domains. It has been proposed that these domains differ in superhelical density. Here, we present evidence that this is probably not the case. A modified Tn10 transposable element was inserted at a number of locations around the E. coli chromosome. This element, mTn10-plac-lacZ⁺, contains the lac operon promoter, plac, whose activity increases with increasing superhelical density, fused to a lacZ⁺ reporter gene. Although mTn10-plac-lacZ⁺ fusion expression varies as much as approximately threefold at different insertion sites, the relative levels of expression from these elements are unaffected by replacing plac with the gyrA promoter, pgyrA, which has a reciprocal response to changes in superhelical density. Importantly, topoisomerase mutations and coumermycin, which inhibits DNA gyrase activity, alter mTn10-plac-lacZ⁺ and mTn10-pgyrA-lacZ⁺ fusion expression in expected ways, showing that the elements remain responsive to supercoiling and that topoisomerase activity is required for maintaining superhelical density. Fusion expression is not affected by anaerobic growth or osmotic shock, two physiological conditions thought to alter supercoiling. The approximately threefold difference in mTn10-plac-lacZ^- and mTn10-pgyrA-lacZ⁺ fusion expression observed at different sites may be explained by regional differences in chromosomal copy number that arise from bidirectional replication. Together, these results strongly suggest that the E. coli chromosomal domains do not differ in functional superhelical density.     

脓肿分枝杆菌MBT结构鉴定与比较演化分析

【背景】作为临床最常见的非结核条件致病分枝杆菌,脓肿分枝杆菌(Mycobacteroides abscessus)因其天然、多耐药等特性成为目前临床治疗的一大挑战。作为分枝杆菌限制性营养元素——铁摄取的关键系统,分枝杆菌素(mycobactin,MBT)、羧基分枝杆菌素(carboxymycobactin,cMBT)与病原分枝杆菌的毒力、耐药等密切相关。【目的】丰富分枝杆菌MBT、cMBT结构数据,探究MBT在致病分枝杆菌起源过程中的演化规律。【方法】在MALDI-TOF-MS与FT-MS/MS解析脓肿MBT、cMBT结构的基础上,进一步开展其活性分析与生物合成基因簇比较基因组分析。【结果】虽然脓肿分枝杆菌MBT、cMBT母核修饰模式与海洋分枝杆菌最相似,R1、R2、R3、R5等位置的修饰完全相同,而且脂肪酸链均位于R4位置;但脂肪酸链长度不同[C10-17 (MBT)、C4-8 (cMBT)],为新结构。Fe-cMBT不仅以浓度依赖方式促进脓肿分枝杆菌生长,而且利用效率显著高于FeCl₃,相关结果表明MBT-cMBT是脓肿分枝杆菌高效获取铁元素的关键系统。与MBT结构结果一致,mbt-1基因簇共线性分析及mbt-1、mbt-2系统发育分析结果均表明脓肿分枝杆菌与海洋分枝杆菌(M.marinum)亲缘关系最近,而非结核分枝杆菌(M.tuberculosis)或耻垢分枝杆菌(M.smegmatis) (基于16S rRNA基因序列分析)。进一步分析发现,M.marinum、M.tuberculosis、M.bovis等病原分枝杆菌脂肪酸链长度变化范围仅4 C,而M.abscessus、M.fortuitum、M.avium和M.smegmatis等条件致病与非致病菌的脂肪酸链长度变化范围为7-11 C,暗示MBT同系物脂肪酸链长度变化范围与分枝杆菌不同生活方式、环境之间可能存在关联。【结论】作为获取铁元素的关键系统,具有独特结构的脓肿分枝杆菌MBT-cMBT在致病、耐药等方面的作用及起源、演化规律值得深入研究。     

Contributions à la réorganisation systématique des Peritelini ouest-paléarctiques. IX. Révision du genre Meira Jacquelin du Val 1852 (Coleoptera : Curculionidae : Entiminae)

The genus Meira Jacquelin du Val 1852 is revised, four lectotypes and a neotype are designated, a new synonymy is proposed and four new species from France – one from Alpes-Maritimes (Meira alpina n. sp.), two from Var (M. echinoides n. sp. and M. teloniensis n. sp.), one from Vaucluse (M. germanni n. sp.) and one from Italy, from Abruzzo (M. osellai n. sp.) are described,. Synonyms, bibliographic and ecological data are provided. Whole-body photos of the types of the old species and of the four new species, drawings of systematically important characters, keys and distribution maps are also presented.     

Interactions of Molecules with Nucleic Acids. X. Covalent Intercalat e Binding of the Carcinogenic BPDE I(+) to Kinked DNA

Abstract

A theoretical model is proposed for the covalent binding of (+) 7 β,8α-dihydroxy-9α, 10α- epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene denoted by BPDE I(+), to N2 on guanine. The DNA must kink a minimum of 39° to allow proper hybrid configurations about the C10 and N2 atoms involved in bond formation and to allow stacking of the pyrene moiety with the non-bonded adjacent base pair. Conservative (same sugar puckers and glycosidic angles as in B-DNA) and non-conservative (alternating sugar puckers as in intercalation sites) conformations are found and they are proposed structures in pathways connecting B-DNA, an intercalation site, and a kink site in the formation of a covalently intercalative bound adduct of BPDE I(+) to N2 on guanine. Stereographic projections are presented for (3′) and (5′) binding in the DNA. Experimental data for bending of DNA by BPDE, orientation of BPDE in DNA and unwinding of superhelical DNA is explained. The structure of a covalent intercalative complex is predicted to result from the reaction. Also, an anti ? syn transition of guanine results in a structure which allows the DNA to resume its overall B-form. The only change is that guanine has been rotated by 200° about its glycosidic bond so that the BPDE I(+) is bound in the major groove. The latter step may allow the DNA to be stored with an adduct which may produce an error in the genetic code.     

Magnolia (Magnoliaceae) in Mexico and Central America: a synopsis

As a result of a recent alpha-taxonomic study of the genusMagnolia s. str. in Mexico and Central America, four new species and four new subspecies ofMagnolia sect.Theorhodon are proposed, includingM. iltisiana, M. panamensis, M. tamaulipana, M. sororum subsp.lutea, andM. pacifica, consisting of three subspecies:pacifica, pugana, andtarahumara. Additionally, a new combination,M. guatemalensis subsp.hondurensis, is made, and a new section,M. sect.Splendentes, is erected for the Caribbean species. The results have implications for understandingMagnolia biogeography, andM. tamaulipana has potential for horticulture in temperate zones. Keys to genera, sections, and species, and maps of species distributions are presented.Magnolia grandiflora L. andM. schiedeana Schltdl. are lectotypified. Como resultado de un estudio alfa-taxonómico reciente del géneroMagnolia s.str. en México y Centro América, se proponen cuatro especies nuevas y cuatro subespecies nuevas deMagnolia sect.Theorhodon, incluyendoM. iltisiana, M. panamensis, M. tamaulipana, M. sororum subsp.lutea y.M. pacifica, la última consiste de tres subespecies:pacifica, pugana ytarahumara. Adicionalmente, se hace la combinaciónM. guatemalensis subsp.hondurensis y se erigeM. sect.Splendentes para las especies del Caribe. Los hallazgos tienen implicaciones en el entendimiento de la biogeografia deMagnolia y.M. tamaulipana tiene potencial en la horticultura de zonas templadas. Se presentan claves para géneros, secciones y especies, y mapas de distribución para las especies.Magnolia grandiflora L. yM. schiedeana Schltdl. son lectotipificadas.     

Circular dichroism of superhelical DNA

The circular dichroism (CD) spectra of a number of superhelical DNA's have been measured. The introduction of negative superhelical turns causes an increase in magnitude of the positive band around 280 mμ, while the trough around 250mμ is little affected. For two samples of λb2b5c DNA (20 Mdalton) containing different number of negative superhelical turns, the magnitude of the positive band relative to that of the nicked control increases with increasing number of superhelical turns. In 2M NaCl, the small (1.45 Mdalton) superhelical DNA from E. coli 15 shows an unusually large difference in CD compared with that of the same DNA with a few single-chain scissions per molecule. This large difference is not observed in a medium containing p. 0.11M NaCl. These results indicate that the double helix in a superhelical DNA is perturbed somewhat due to the bending and torsional forces in such a molecule. The magnitude of such structural alteration seems to depend on the number of superhelical turns per unit length, the size of the DNA molecule, as well as the ionic medium.     

Studies on deoxyribonucleoprotein structure. Small-angle X-ray scattering in solutions of various ionic strengths.

The cross-sectional radius of gyration of the deoxyribonucleoprotein (DNP) threads was measured by small-angle X-ray scattering in a wide range of ionic strengths (from 0.0005 to 2 M NaCl). For DNP in a solution of low ionic strength, this value is 30 Å. The increase of ionic strength results in partial deproteinization of DNP, while the cross-sectional radius of gyration varies from 25 Å for DNP in 0.7 M NaCl to 10 Å for DNP in 2 M NaCl. It is suggested that gradual deproteinization by the increase of NaCl concentration causes conformational changes, which are associated with the alteration of the DNP superstructure. The data are interpreted on the basis of the superhelical model of DNA packing in DNP; however, the coexistence of superhelical and unfolded regions in the DNP structure is also a possibility.     

Design and synthesis of naphthalimide group-bearing thioglycosides as novel β-N-acetylhexosaminidases inhibitors

GH20 human β-N-acetylhexosaminidases (hsHex) and GH84 human O-GlcNAcase (hOGA) are involved in numerous pathological processes and emerged as promising targets for drug discovery. Based on the catalytic mechanism and structure of the catalytic domains of these β-N-acetylhexosaminidases, a series of novel naphthalimide moiety-bearing thioglycosides with different flexible linkers were designed, and their inhibitory potency against hsHexB and hOGA was evaluated. The strongest potency was found for compound 15j (K_i?=?0.91?µM against hsHexB; K_i?>?100?µM against hOGA) and compound 15b (K_i?=?3.76?µM against hOGA; K_i?=?30.42?µM against hsHexB), which also exhibited significant selectivity between these two enzymes. Besides, inhibitors 15j and 15b exhibited an inverse binding patterns in docking studies. The determined structure–activity relationship as well as the established binding models provide the direction for further structure optimizations and the development of specific β-N-acetylhexosaminidase inhibitors.     

New data on the Merodon Meigen 1803 fauna (Diptera: Syrphidae) of Turkey including description of a new species and changes in the nomenclatural status of several taxa

Abstract

The Old World syrphid genus Merodon Meigen 1803 is highly species-diverse and has a significant number of endemic species in the circum-Mediterranean area. The present study reports on taxonomic changes resulting from the examination of adult Merodon specimens collected in 15 Turkish provinces during the period 1992 to 2002, and provides new faunistic data. Merodon ilgazense n. sp. is described. Four species of Merodon new to Turkey are recorded: M. armipes Rondani 1843, M. auronites Hurkmans 1993, M. bessarabicus Paramonov 1924 and M. chalybeatus Sack 1913. Lectotypes are designated for two taxa: M. chalybeatus Sack 1913 and M. clunipes Sack 1913. Merodon italicus Rondani 1845 rev. stat. is reinstated as a valid species. Following a detailed study of the type material in different entomological collections, the status of 10 taxa is revised and five new synonymies are proposed: M. albonigrum Vuji Radenkovi &; S?imi 1996 n. syn. (=junior synonym of M. chalybeatus Sack 1913); M. alexeji Paramonov 1925 n. syn. (=junior synonym of M. serrulatus Wiedemann in Meigen 1822); M. aureotibia Hurkmans 1993 n. syn. (=junior synonym of M. vandergooti Hurkmans 1993); M. kaloceros Hurkmans 1993 n. syn. (=junior synonym of M. erivanicus Paramonov 1925); M. longicornis Sack 1913 n. syn. (=junior synonym of M. italicus Rondani 1845).     

A Structural Transition in d(AT) n ·d(AT) n Inserts within Superhelical DNA

Abstract

We have constructed plasmids carrying d(AT)_n·d(AT)_n inserts of different lengths. Two- dimensional gel electrophoresis patterns show that an increase in the negative superhelicity of these DNAs brings about a structural transition within the inserts, resulting in a reduction of the superhelical stress. However, this reduction corresponds to the expected values neither for cruciform nor for the Z form. Those DNA topoisomers in which the structural transition had occurred proved to be specifically recognizable by single-strand-specific endonuclease SI, with the cleavage site situated at the centre of the insert. These data, as well as kinetic studies, suggest that the cloned d(AT)_n·d(AT) _n sequences adopt a cruciform rather than the Z-form structure. We discuss plausible reasons of the discrepancy between the observed superhelical stress release and that expected for the transition of the insert to the cruciform state.     

Effect of Novel Amino Acids and Dipeptides Substituted 3-Morpholino Arecoline Derivatives as Muscarinic Receptor 1 Agonists in Alzheimer’s Dementia Models

A series of novel, potent and selective muscarinic receptor 1 agonists (M1 receptor agonists) that employ a key N-substituted morpholine arecoline moiety has been synthesized as part of research effort for the therapy of Alzheimer’s diseases. The ester group of arecoline (which is reported as mucarinic agonist) has been replaced by N-substituted morpholine ring. The structure activity relationship reveals that the increase in lipophilic carbon chain on the nitrogen atom of the morpholine ring increases the affinity of M1 receptor. In the present study, we are reporting N-amino acid substituted 9(a–k) and dipeptides substituted 10(a–j) and 11(a–j) morpholino arecoline derivatives, along with their in vitro muscarinic binding studies by using [³H]QNB and also in vivo evaluation of memory and learning in male Wistar rats (passive avoidance test plus maze studies) as M1 receptor agonist. Some molecules from the dipeptide series (10b, 10c and 10j) showed potent M1 receptor agonist activity. Other derivatives also showed considerable M1 receptor binding affinity.     

Dynamic light scattering as a probe of superhelical DNA-intercalating agent interaction

Polarized dynamic light-scattering measurements on superhelical pBR322-plasmid DNA solutions in 0.2M NaCl, 2 mM NaPi, pH 7.0, 2 mM EDTA result in a translational diffusion coefficient D = (3.77 ± 0.10) × 10^?8 cm²/s for the native molecule. Modeling the DNA, in the simplest approximation, as a 10 × 440-nm effective hydrodynamic rigid rod yields a good fit to the apparent diffusion coefficient angular-dependence data up to 70°; the model fails at higher angles, probably due to the effects of flexibility or branching of the rod. Diffusion coefficient titration experiments with a platinum complex intercalating agent (PtTS) result in a titratable superhelix density of σ = ?0.079 ± 0.008 under our experimental conditions, corresponding to about 34 superhelical turns in the native DNA. The DNA contour length predicted by our two independent results, the rod dimensions and the number of superhelical turns, is in excellent agreement with the contour length calculated from the number of base pairs, supporting the hydrodynamic approximation of an effective rodlike structure for this small DNA molecule in solution.