Echinomycin binding sites on DNA

The preferred binding sites of echinomycin on DNA can be determined by a method called "footprinting." A 32P end-labeled restriction fragment from pBR322 DNA is protected by binding to echinomycin, and cleaved by a synthetic DNA cleaving reagent, methidiumpropyl--EDTA . Fe(II); the DNA cleavage products are then subjected to high-resolution gel analyses. This method reveals that echinomycin has a binding site size of four base pairs. The strong binding sites for echinomycin contain the central two-base-pair sequence 5'-CG-3'. From an analysis of 15 echinomycin sites on 210 base pairs of DNA, key recognition elements for echinomycin are contained in the sequences (5'-3') ACGT and TCGT (A, adenine; C, cytosine; G, guanine; T, thymine).     

Site-specific cleavage of a yeast chromosome by oligonucleotide-directed triple-helix formation

Oligonucleotides equipped with EDTA-Fe can bind specifically to duplex DNA by triple-helix formation and produce double-strand cleavage at binding sites greater than 12 base pairs in size. To demonstrate that oligonucleotide-directed triple-helix formation is a viable chemical approach for the site-specific cleavage of large genomic DNA, an oligonucleotide with EDTA-Fe at the 5' and 3' ends was targeted to a 20-base pair sequence in the 340-kilobase pair chromosome III of Saccharomyces cerevisiae. Double-strand cleavage products of the correct size and location were observed, indicating that the oligonucleotide bound and cleaved the target site among almost 14 megabase pairs of DNA. Because oligonucleotide-directed triple-helix formation has the potential to be a general solution for DNA recognition, this result has implications for physical mapping of chromosomes.     

猪羊生长激素基因保守性研究

生长激素基因在猪羊间的差异 ,通过两对引物 ,分别为P1:5’ -agctggattctttacggctgagcc - 3’和P2 :5’ -tccggaagcaggagagcagaccgtag - 3’ ,P3:5’ -gctgacacctacaaggagtttg - 3’和P4:5’ - gtttgcttgaggatctgtcctg - 3’。对它们的DNA进行聚合酶链反应 ,电泳及序列分析结果表明 :P1-P2和P3-P4引物都能扩增出猪羊DNA样 ,得到大小一致的扩增带 (对应引物的带 ) ,但southern杂交结果的差别很大 ;生长激素基因的碱基组成在猪羊间的差异为 6 5 % ,即猪羊生长激素基因保守性很强     

The DNA-binding properties of the major regulatory protein alpha 4 of herpes simplex viruses

The transition from the expression of alpha, the first set of five herpes simplex virus genes expressed after infection, to beta and gamma genes, expressed later in infection, requires the participation of infected cell protein 4 (alpha 4), the major viral regulatory protein. The alpha 4 protein is present in complexes formed by proteins extracted from infected cells and viral DNA fragments derived from promoter domains. This report shows that the alpha 4 protein forms specific complexes with DNA fragments derived from 5' transcribed noncoding domains of late (gamma 2) genes whose expression requires viral DNA synthesis as well as functional alpha 4 protein. Some of the DNA fragments to which alpha 4 binds do not contain homologs of the previously reported DNA binding site consensus sequence, suggesting that alpha 4 may recognize and interact with more than one type of DNA binding site. The alpha 4 proteins can bind to DNA directly. A posttranslationally modified form of the alpha 4 protein designated alpha 4c differs from the alpha 4a and alpha 4b forms with respect to its affinity for DNA fragments differing in the nucleotide sequences of the binding sites.     

The Prp19p-associated complex in spliceosome activation

During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.     

生物素标记犬瘟热病毒DNA探针的制备与纯化

用以选择犬瘟热病毒ＲＮＡ的有关碱基顺序，设计ＤＮＡ探针，并在５＇末端偶关一个氨基连接分子：５×ＡＧＧＧＣＴＣＡ－ＧＧＴＡＧＴＣＣＡＧＣＡＡＴＧ３＇，共２３个碱基和一个氨基连接分子。ＮＨ２－ＤＮＡ探针在ＤＮＡ合成仪上合成。合成产物同生物素酰氨基已酸盐Ｎ－羟琥珀酰亚胺酯反应，反应物经高效硅胶薄层板纯化，得到生物素标记犬温热病毒ＤＮＡ探针。     

Recognition of thymine adenine.base pairs by guanine in a pyrimidine triple helix motif

Oligonucleotide recognition offers a powerful chemical approach for the sequence-specific binding of double-helical DNA. In the pyrimidine-Hoogsteen model, a binding size of greater than 15 homopurine base pairs affords greater than 30 discrete sequence-specific hydrogen bonds to duplex DNA. Because pyrimidine oligonucleotides limit triple helix formation to homopurine tracts, it is desirable to determine whether oligonucleotides can be used to bind all four base pairs of DNA. A general solution would allow targeting of oligonucleotides (or their analogs) to any given sequence in the human genome. A study of 20 base triplets reveals that the triple helix can be extended from homopurine to mixed sequences. Guanine contained within a pyrimidine oligonucleotide specifically recognizes thymine.adenine base pairs in duplex DNA. Such specificity allows binding at mixed sites in DNA from simian virus 40 and human immunodeficiency virus.     

火炬松木质素合成中CAD基因单核苷酸多态性检测

该研究以美国南部重要的造纸工业用材树种火炬松为材料 ,利用Transgenomic公司最近推出的Wave DNA片段分析系统 ,快速检测肉桂醇脱氢酶 (CAD)基因在个体间的单核苷酸多态性 ,获得了令人满意的结果 .首先利用Oligo5 .1软件对火炬松CAD基因全序列分区域进行引物设计 ,当设定每一区域能扩增出 10 0～ 2 0 0bp产物时 ,共在 8个碱基区域 (S1～S8)分别设计出了最佳正向引物和反向引物 ;以来自火炬松 12株优树自由授粉种子的胚乳中提取的DNA为材料 ,对CAD基因 8个碱基区域分别进行PCR扩增 ,有 5个碱基区域 (S3 ,S5 ,S6 ,S7,S8)得到了唯一的并且与预期碱基长度基本一致的PCR扩增产物 ,对其余 3个碱基区域进一步进行FailSafeTM PCR扩增 ,它们均在某些PCR缓冲液中得到了唯一的扩增产物 ;利用Wave DNA片段分析系统 ,对 6个碱基区域 (S2 ,S3,S5 ,S6 ,S7,S8)进行单核苷酸多态性检测 ,结果表明来自优树 2 2的种子与其它 11株优树的种子之间存在碱基序列变异 ,其中在S5 ,S6区域检测到了较大的单核苷酸多态性 ,这两个区域分别位于CAD基因的第 2和第 3内含子 (Intron2 ,Intron3) .