Binding of Non-Intercalating Antibiotics to B-DNA: A Theoretical Study Taking Into Account Nucleic Acid Flexibility

Abstract

A detailed theoretical study has been made for five antibiotics which all bind selectively to AT sequences in the minor groove of B-DNA: SN-18071, NSCT-101327, distamycin-2, distamycin-3 and netropsin. The optimal complexes were found for systems in which the flexibility of DNA, as well as that of the antibiotics, was taken into account. Explicit, mobile counterions and a dielectric function modelling aqueous solution were also included. The binding geometries of the most strongly interacting antibiotics, distamycin-3 and netropsin, are compared in considerable detail and it is shown that notable differences exist between them. The results for netropsin are also discussed in the light of recent disagreements concerning its exact binding location within DNA.     

北京鸭线粒体基因组全序列测定和分析

线粒体DNA作为遗传标记,已在家鸡(Gallus gallus)和家鹅(Anser anser)的研究中取得了重大进展,而对家鸭(Anas platyrhychos domesticus)的研究却很少.本研究参照近源物种线粒体基因组序列设计15对引物,通过PCR扩增、测序、拼接,获得北京鸭(A.platyrhychos)线粒体基因组全序列,初步分析其特点和各基因的定位.结果显示,北京鸭线粒体基因组全长16 604 bp,碱基组成为29.19%A、22.20%T、15.80%G、32.81%C,包含13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码控制区(D-loop),基因组成及排列顺序与其他鸟类相似.基于线粒体D-loop区全序列,用N-J法构建了7种雁形目鸟类系统进化树,结果表明,北京鸭与绿头鸭(A.platyrhychos)系统进化关系较近.     

Relative Stabilities and Transitions of DNA Conformations in 1:1 Electrolytes: A Theoretical Study

Abstract

We use a recently developed formalism (1) to calculate the salt dependent part of the free energy determining DNA conformational stability in 1:1 electrolytes. The conformations studied are the A,B,C and alternating-B right-handed forms and the Z₁Z_II left-handed forms of DNA. In the case of the B-Z₁ transition of d(G-C) · d(G-C) helices in NaCl solution, the free energy contribution considered suffices to describe the transition in a quantitative manner. The theory also predicts the occurrence of salt-induced B-A transitions which have been recently observed with poly[d(n²A-T)| and poly[d(G-C)|. In other cases, additional terms in the free energy balance, particularly due to hydration effects, must be at least as important as salt effects in determining conformational stability and structural transitions in solution. If diffuse ionic cloud electrostatic effects alone would dominate in all cases, the relative helical stabilities at 0.2 M monovalent salt would decrease in the order C > B > A > Z_II > Z₁ > alternating-B. At high salt concentrations (2.0 M - 5.0 M), the order would be alternating-B > Z, > A > Z_II > B > C.     

A Theoretical Investigation on the Sequence Selective Binding of Daunomycin to Double-Stranded Polynucleotides

Abstract

Theoretical computations are performed on the structural and energetical factors involved in the sequence selective binding of daunomycin (DNM) to six representative self- complementary double-stranded hexanucleotides: d(CGTACG)₂, d(CGATCG)₂, d(CITACI)₂, d(TATATA)₂, d(CGCGCG)₂ and d(TACGTA)₂. The conformational angles of the hexanucleotides are fixed in values found in the representative crystal structure of the d(CGTACG)₂- DNM complex. The intermolecular DNM-hexanucleotide interaction energies and the conformational energy changes of DNM upon binding are computed and optimized in the framework of the SIBFA procedure, which uses empirical formulas based on ab initio SCF computations. Among the two regularly alternating hexanucleotides, d(TATATA)₂ and d(CGCGCG)₂, a stronger binding is predicted for the former, in agreement with experimental results obtained with poly(dA-dT)-poly(dA-dT) and poly(dG-dC)<<poly(dG-dC). Altogether, however, among the six investigated sequences, the strongest complexes are computed for the mixed hexanucleotides d(CGATCG)₂ and d(CGTACG)₂, containing the intercalation site between two CG base pairs and an adjacent TA base pair. This situation may be related to the increased affinity of DNM for GC rich DNA's and to the situation in the crystal structure of the DNM-d(CGTACG)₂ complex. Analysis of the intrinsic base sequence preferences expressed by the individual constituents of DNM, namely the daunosamine side chain, the chromophore ring and its two 9-hydroxyl and 9-acetoxy substituents, reveals that the overall sequence preference found is the result of a rather intricate interplay of intrinsic sequence preferences, in particular at the level of daunosamine and the 9-hydroxyl substituent. Altogether, it is seen that the selective base pair recognition by daunomycin cannot, in general, be defined in terms of the two base pairs implicated in the intercalation site alone (with the exception of homogeneous AT or GC base sequences) but must be expressed in terms of a triplet of base pairs.     

中缅树鼩UCP2 cDNA核心片段的序列分析

解偶联蛋白2(UCP2)是近年新发现的一种解偶联蛋白,具有多种生物学活性,相关研究表明,UCP2可以抑制某些细胞(如免疫细胞等)线粒体活性氧的过量生成.本研究通过设计简并引物进行RT-PCR从中缅树鼩(Tupaia belangeri肝中获得UCP2基因cDNA核心序列.该片段长745 bp,推测氨基酸序列为248个氨基酸.结构功能分析发现,此段氨基酸序列具有2个线粒体内膜载体蛋白特征结构、5个跨膜α-螺旋结构域、1个嘌呤结合区域(PNBD)以及3个解耦联蛋白质的特征序列.中缅树鼩UCP2氨基酸序列与普氏野马(Equus caballus)、小家鼠(Mus musculus)、家犬(Canis lupus familiaris)、人(Homo sapiens)、褐家鼠(Rattus norvegicus)、豚鼠(Cavia porcellus)、苏门达腊猩猩(Pongo abelii)、加卡利 安鼠(Phodopus sungorus)和马铁菊头蝠(Rhinolophus ferrumequinum)UCP2进行比较,氨基酸同源性均在90％以上.同时,本研究通过MEGA5构建系统树,对UCP2分子进化特征进行了一定的探讨.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

鹟科五种鸟线粒体DNA序列变化与亲缘关系的研究

首次对鹟科（Muscicapidae）画眉亚科三种鸟----画眉（Garrulax canorus）、红嘴相思鸟（Leiothrix lutea）、棕头雅雀(Paradoxornis webbiana);鸫亚科的乌鸫（Turdinae merula）线粒体DNA的12S rRNA基因片段的DNA序列进行了测定,并与北美画眉亚科弯嘴鹛属（Pomatostomus）的灰冠弯嘴鹛（P.temporalis）同源序列进行分析比较并构建分子进化树。实验结果与传统形态学论述存在一定的差异,与同工酶研究结果相同：画眉与红嘴相思鸟的亲缘关系最近,棕头鸦雀较乌鸫与画眉亚科的关系更近,为将其置于画眉亚科提供了分子证据。 Abstract：The classfication on Muscicapinae disturbed us for a long time.The important thing is to find an appropriate character to resolve it.It is the first time that we testified the mitochondrial 12S rRNA gene fragment sequence of four birds (G.canorus,L.lutea,P.webbianus and T.merula-owing to Muscicapinae) and aligned with the same sequence of P.temporalis.Using the mitochondrial 12S rRNA sequences date,we built phylogenetic trees.The results indicated that that the relationship between Garrulax canorus and Leiothrix lutea in the four birds was closer which is congruent with morphology.According to the results of isoenzyme and DNA sequencing data,we can draw a conclusion that Paradoxornis webbianus has closer relationship to Timaliinae than that of Turdus merula,and that putting Paradoxonithinae to Timaliinae was reasonable.     

Characterization and phylogenetic analysis of the complete mitogenome of Allactaga sibirica (Rodentia: Dipodidae)

Allactaga sibirica (Dipodidae) is widely distributed in the northwestern arid regions of China. The complete mitochondrial genome (mitogenome) of A. sibirica was 16,685 bp in length; included 13 protein-coding genes, 2 ribosome genes, 22 tRNA genes, and one control region; and had a structure that was typical of vertebrates. The base composition and codon usage of the mitogenome are described, and the structure of the non-coding sequence in the A. sibirica is reported for the first time. The putative origin of replication for the light strand of A. sibirica was approximately 45 bp long, and was highly conserved in the stem-loop and adjacent sequences. Phylogenetic analyses showed high resolution in each of the main divergent clades within Dipodoidea using mitogenomes data. The results indicated that the Zapodidae group was a representative of very basal taxon in Dipodoidea, and shared a common ancestor with Dipodidae species. Within Dipodidae clade, Allactaginae species was at basal position, and this result was in line with previous molecular systematic and morphological studies. Furthermore, Euchoreutes naso and A. sibirica had a close relationship could implicate a sister-group relationship between Euchoreutinae and Allactaginae. Meanwhile, this work also provided a set of useful data on phylogeny and molecular evolution in Dipodidae species.     

TTV武汉分离株DNA克隆和序列分析

用PCR技术从一武汉病人血清中分离获得TGV DNA片断,把此DNA片断克隆到pMD18-T质粒载体中,进行全序列测定并用计算机软件对核苷酸,氨基酸序列和ORF2多肽特征进行比较和分析。所分离获得的TTV DNA片断全长1333bp,含TTV完整的ORF2及部分ORF1序列,核苷酸和氨基酸序列与其它基因型为1a的TTV分离株具有极高的同源性。ORF2多肽含202个氨基酸,在N端部分和C端部分具有较高的亲水性和较强的抗原性,而中间为一段疏水区域,抗原性较弱。N端结构以α螺旋为主,含有典型的酪氨酸激酶磷酸化价点（RARDWYPGY,38－45aa）和三个潜在的蛋白质激酶C的磷酸化位点,C端富含脯氨酸。结构以β转向为主,含有三个连续的N－十四烷酰化位点,推测ORF2编码的蛋白可能是一种磷酸化蛋白。     

7-Azido-Actinomycin D: A Photoaffinity Probe of the Sequence Specificity of DNA Binding by Actinomycin D

Abstract

Actinomycin D (ActD) is a DNA-binding antitumor antibiotic that appears to act in vivo by inhibiting RNA polymerase. The mechanism of DNA binding of ActD has attracted much attention because of its strong preference for 5′-dGpdC-3′ sequences. Binding is thought to involve intercalation of the tricyclic aromatic phenoxazone ring into a GC step, with the two equivalent cyclic pentapeptide lactone substituents lying in the minor groove and making hydrogen bond contacts with the 2-amino groups of the nearest neighbor guanines. Recent studies have indicated, however, that binding is also influenced by next-nearest neighboring bases. We have examined this higher order specificity using 7-azido-actinomycin-D as a photoaffinity probe, and DNA sequencing techniques to quantitatively monitor sites of covalent photoaddition. We found that GC doublets were strongly preferred only if the 5′- flanking base was a pyrimidine and the 3′-flanking base was not cytosine. In addition we observed a previously unreported preference for binding at a GG doublet in the sequence 5′- TGGG-3′.     

Molecular Cloning and Sequence Analysis of a Gene Encoding Rice 10 kD Prolamin

Using PCR technique, two prolamin genes from Oryza sativa var. indica (cv. Guanglu′ ai) and O. sativa var. japonica (cv. Zhonghua 8) were amplified and cloned. The prolamin gene contained 525 base pairs and encoded 134 amino acid residues. The two genes cloned from two different rice cultivars exhibited 100% homology and were highly homologous with the 10 kD prolamin gene in other rice species amountin an homology ranging from 96.6% to 100%. The deduced amino acid sequence shared 34.2% homology with that of maize 10 kD prolamin. As for dicots, only two types of storage protein shared some homology with rice 10 kD prolamin. One was from Brazil nut and the other from castor bean. Analysis on the signal peptide of rice 10 kD prolamin showed that it shared higher homology with that of storage proteins in some monocots such as maize, sorghum and oat. No similar sequence was found in dicots. The gene sequences of "Guangluai” and "Zhonghua 8” 10 kD prolamin would appear in EMBL data-base under the accession number L36604 and L36605 respectively.     

核糖体DNA序列分析在昆虫系统学研究中的应用

随着分子生物学技术的迅速发展,DNA序列分析在昆虫系统学研究中的应用不断增多。本文系统阐述了核糖体DNA的结构、分类学意义、以及在昆虫不同类群不同阶元之间系统发育关系研究中的应用,概要介绍了序列分析方法,并对其应用前景进行了展望。     

平胸龟Sox基因的克隆和序列分析

本文采用ＰＣＲ技术扩增和克隆了平胸龟的Ｓｏｘ基因,并通过银染测序进行了序列分析。结果显示,平胸龟雌雄个体均能扩增出相同长度的片段,其ＤＮＡ序列与人ＳＲＹ基因的ＨＭＧ－ｂｏｘ同源性达７２．７％,推测的氨基酸序列与ＳＲＹ基因的ＨＭＧ－ｂｏｘ序列同源性为４６．５％,充分显示出Ｓｏｘ基因在系统进化上的保守性。本文为Ｓｏｘ基因的起源和进化研究提供了资料。     

三种海豚线粒体COI基因的序列分析

对瓶鼻海豚(Tursiops truncatus)、中华白海豚(Sousa chinensis)和糙齿海豚(Steno bredanensis)的线粒体DNA COI基因进行了测序分析。PCR产物约700bp。扩增产物直接测序,去除引物序列后分别获得643、618和618bp的核苷酸序列。碱基组成平均为,T:31·07%,C:26·13%,A:27·27%,G:15·50%,GC含量为41·63%,其中碱基G的含量明显较低。与Gen Bank中9种鲸的同源序列比对,去除部分端部序列后得到597个比对位点,包括141个简约信息位点,43个单突变子,无插入/缺失位点。12种鲸的种间序列差异较大,其序列变异度在2·1%～17·1%之间。597个比对位点编码199个氨基酸,其中有9个氨基酸发生改变,其中一个氨基酸突变可将齿鲸亚目和须鲸亚目分开。NJ系统树表明,海豚科形成单系类群,瓶鼻海豚和中华白海豚的亲缘关系较近。上述分析表明,COI基因可用于鲸类的种类鉴定和系统发育分析。     

我国森林脑炎病毒森张株编码区序列测定

为鉴定我国森林脑炎病毒亚型,了解基因组结构与生物功能的关系,同时为森林脑炎病毒新型疫苗研制打下基础,对森林脑炎病毒森张株编码区序列进行测定。根据已发表的Sofjin-HO、Oshima5-10株序列设计9对重叠引物,通过RT—PCR扩增不同的cDNA片段,分别克隆至pGEM—T载体,转化DH5α菌,挑阳性克隆PCR、酶切鉴定后测序。结果表明森张株编码区全长10245nt,编码3414个氨基酸。我国森林脑炎病毒森张株与Oshima5—10、Sofiin—HO、Sofiin同源性最近,属于远东亚型。     

中国地鼠线粒体基因组的序列分析与分子进化

目的 获得中国地鼠线粒体基因组序列,为线粒体疾病模型提供分子数据.方法 参照近缘物种的线粒体基因组序列,设计27对特异引物,采用TD-PCR及测序技术获得了中国地鼠的线粒体全基因组序列,分析了其基因组特点和各基因的定位.还结合GenBank中已发表的其他5种啮齿类动物的线粒体基因组序列,探讨啮齿类动物不同科间的系统进化关系.结果 中国地鼠线粒体基因组全长为16 283 bp,碱基组成为33.53％A、30.50％T、12.98％G、22.80％C,包括13个蛋白质编码基因、2个rRNA基因、22个tRNA基因和1个非编码基因控制区.中国地鼠和金黄地鼠亲缘关系最近.结论 中国地鼠线粒体基因组各基因长度、位置与典型的啮齿类动物相似,其编码蛋白质区域和rRNA基因与其他啮齿类动物具有很高的同源性,显示线粒体基因组在进化上十分保守.5种动物的分子系统进化树与传统分类地位一致.     

Theoretical Investigation on the Binding Specificity of Sialyldisaccharides with Hemagglutinins of Influenza A Virus by Molecular Dynamics Simulations

Recognition of cell-surface sialyldisaccharides by influenza A hemagglutinin (HA) triggers the infection process of influenza. The changes in glycosidic torsional linkage and the receptor conformations may alter the binding specificity of HAs to the sialylglycans. In this study, 10-ns molecular dynamics simulations were carried out to examine the structural and dynamic behavior of the HAs bound with sialyldisaccharides Neu5Acα(2–3)Gal (N23G) and Neu5Acα(2–6)Gal (N26G). The analysis of the glycosidic torsional angles and the pair interaction energy between the receptor and the interacting residues of the binding site reveal that N23G has two binding modes for H1 and H5 and a single binding mode for H3 and H9. For N26G, H1 and H3 has two binding modes, and H5 and H9 has a single binding mode. The direct and water-mediated hydrogen bonding interactions between the receptors and HAs play dominant roles in the structural stabilization of the complexes. It is concluded from pair interaction energy and Molecular Mechanic-Poisson-Boltzmann Surface Area calculations that N26G is a better receptor for H1 when compared with N23G. N23G is a better receptor for H5 when compared with N26G. However, H3 and H9 can recognize N23G and N26G in equal binding specificity due to the marginal energy difference (≈2.5 kcal/mol). The order of binding specificity of N23G is H3 > H5 > H9 > H1 and N26G is H1 > H3 > H5 > H9, respectively. The proposed conformational models will be helpful in designing inhibitors for influenza virus.     

纤毛鹅观草DREB类基因RcDREB1的克隆及其蛋白结合干旱应答元件DRE的功能验证

以纤毛鹅观草为材料,通过RT-PCR和RACE技术,作者首次克隆了一个纤毛鹅观草DREB类基因的全长cDNA序列,命名为RcDREB1。该基因编码蛋白含有一个保守的AP2结构域,表明该基因是AP2大家族的一个新成员。种系发生树分析表明,RcDREB1与小麦AP2家族DREB类基因高度同源。酵母单杂交试验表明,RcDREB1表达的蛋白具有特异结合干旱应答DRE元件的功能;通过实时定量PCR分析发现,高盐、干旱、重金属镉和低温胁迫均可诱导RcDREB1表达,但其对高盐反应较为显著。本研究表明,RcDREB1是DREB类转录因子基因,在介导植物响应逆境信号方面可能发挥着重要生物学功能,为进一步分析小麦族的进化和转基因应用奠定了基础。     

Expression and Sequence Analysis of a cDNA Relative to Orchid Ovule Development

The Phalaenopsis sp. cv. SM 9108 flower provides a good system to isolate ovule-specific genes. A cDNA library at mature ovule stage has been constructed. A differential screening approach was used to identify cDNAs representing genes which are expressed in a stage-specific manner during ovule development. The authors have demonstrated that the expression of a cDNA (0138) was regulated stage-specifically and tis-sue-specifically using, Northern blot, and also have analyzed the full sequence of this cDNA. Its further functional characterization in ovule development will be facilitated.     

A Cladistic Analysis of the Evolutionary Relationships of the Members of the Tyrosinase Gene Family Using Sequence Data

Recently, DNA sequence data have been published on tyrosinase and tyrosinase-related proteins (TRPs) in a wide variety of vertebrates ranging from Rana to Homo. These proteins are in turn members of a larger family of binuclear copper-binding proteins, which all contain two highly conserved copper-binding domains. This gene family also includes tyrosinases from fungi and bacteria as well as arthropodan and molluscan hemocyanins. Parsimony-based alignment and tree construction algorithms (Malign, vl.85 and PAUP, 3.1.1) were used to analyze the diversification of both the evolutionarily conserved copper-binding domains i6n copper-binding proteins in general as well as the diversification of the vertebrate tyrosinase gene family more specifically. These analyses show that the diversification of the vertebrate tyrosinase gene family minimally predates the diversification of vertebrates. Vertebrate tyrosinases proper first diverged from an ancestral tyrosinase-related protein (TRP) that then subsequently diverged to form tyrosinase-related protein-Is (TRP-1s) and tyrosinase-related protein-2s (TRP-2s).