Structure of the DNA-Eco RI endonuclease recognition complex at 3 A resolution

The crystal structure of the complex between Eco RI endonuclease and the cognate oligonucleotide TCGCGAATTCGCG provides a detailed example of the structural basis of sequence-specific DNA-protein interactions. The structure was determined, to 3 A resolution, by the ISIR (iterative single isomorphous replacement) method with a platinum isomorphous derivative. The complex has twofold symmetry. Each subunit of the endonuclease is organized into an alpha/beta domain consisting a five-stranded beta sheet, alpha helices, and an extension, called the "arm," which wraps around the DNA. The large beta sheet consists of antiparallel and parallel motifs that form the foundations for the loops and alpha helices responsible for DNA strand scission and sequence-specific recognition, respectively. The DNA cleavage site is located in a cleft that binds the DNA backbone in the vicinity of the scissile bond. Sequence specificity is mediated by 12 hydrogen bonds originating from alpha helical recognition modules. Arg200 forms two hydrogen bonds with guanine while Glu144 and Arg145 form four hydrogen bonds to adjacent adenine residues. These interactions discriminate the Eco RI hexanucleotide GAATTC from all other hexanucleotides because any base substitution would require rupture of at least one of these hydrogen bonds.     

Location of the c-yes gene on the human chromosome and its expression in various tissues

Analysis of DNA from human embryo fibroblasts showed that ten Eco RI fragments were hybridizable with the Yamaguchi sarcoma virus oncogene (v-yes). Four of the Eco RI fragments were assigned to chromosome 18 and one to chromosome 6. There was evidence for multiple copies of yes-related genes in the human genome; however, only a single RNA species, 4.8 kilobases in length, was related to yes in various cells.     

伪狂犬病病毒gG基因缺失通用转移载体的构建

对克隆有伪狂犬病病毒Ea株基因组DNA SphI 15kb片段的质粒pBSA用SphI和KpnI消化，将含gG全基因及其上游PK基因，下游gD基因部分编码区的约2．6kb的片段亚克隆到消除了EcoRI位点的载体pUC19中，获得重组质粒pUSK(E)。以pEGFP-N1为模板，通过PCR扩增约0．3kb的SV40Poly(A)片段，并将其克隆到杆状病毒转座载体pFastBac1的NotI和PstI位点，利用pFastBac1的BamHI和PstI将含有9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区约9个酶切位点以及SV40Poly(A)的片段亚克隆到pUSK(E)的相应位点，在插入的同时导致gG基因5‘端编码区的400bp的缺失，构建了由gG启动子驱动gG部分编码区缺失的通用转移载体pgG-Uni。序列分析进一步证实：有7个单一酶切位点可供外源基因直接插入。上述结果为构建以伪狂犬病病毒作载体的多价基因工程疫苗以及利用标记蛋白探讨伪狂犬病病毒在体内的增殖与分布奠定了基础。     

Selective cleavage of human DNA: RecA-assisted restriction endonuclease (RARE) cleavage

Current methods for sequence-specific cleavage of large segments of DNA are severely limited because of the paucity of possible cleavage sites. A method is described whereby any Eco RI site can be targeted for specific cleavage. The technique is based on the ability of RecA protein from Escherichia coli to pair an oligonucleotide to its homologous sequence in duplex DNA and to form a three-stranded complex. This complex is protected from Eco RI methylase; after methylation and RecA protein removal, Eco RI restriction enzyme cleavage was limited to the site previously protected from methylation. When pairs of oligonucleotides are used, a specific fragment can be cleaved out of genomes. The method was tested on lambda phage, Escherichia coli, and human DNA. Fragments exceeding 500 kilobases in length and yields exceeding 80 percent could be obtained.     

The energetic basis of specificity in the Eco RI endonuclease--DNA interaction

High sequence selectivity in DNA-protein interactions was analyzed by measuring discrimination by Eco RI endonuclease between the recognition site GAATTC and systematically altered DNA sites. Base analogue substitutions that preserve the sequence-dependent conformational motif of the GAATTC site permit deletion of single sites of protein-base contact at a cost of +1 to +2 kcal/mol. However, the introduction of any one incorrect natural base pair costs +6 to +13 kcal/mol in transition state interaction energy, the resultant of the following interdependent factors: deletion of one or two hydrogen bonds between the protein and a purine base; unfavourable steric apposition between a group on the protein and an incorrectly placed functional group on a base; disruption of a pyrimidine contact with the protein; loss of some crucial interactions between protein and DNA phosphates; and an increased energetic cost of attaining the required DNA conformation in the transition state complex. Eco RI endonuclease thus achieves stringent discrimination by both "direct readout" (protein-base contracts) and "indirect readout" (protein-phosphate contacts and DNA conformation) of the DNA sequence.     

Human c-Ki-ras2 proto-oncogene on chromosome 12

A human colonic adenocarcinoma transforming gene, recently identified as a cellular homolog of the Kirsten sarcoma gene (v-ras), was used to assign the human cellular Kirsten ras2 gene to chromosome 12 by the Southern hybridization method. A single 640 base-pair Eco RI--Hind III fragment of the transforming gene, isolated by DNA transfection and molecular cloning, can detect a single Eco RI fragment (2.9 kilobase pairs) of DNA from phenotypically normal cells. The data suggest a constant chromosomal location of c-Ki-ras2.     

Properties of a normal mouse cell DNA sequence (sarc) homologous to the src sequence of Moloney sarcoma virus

A 15.0-kilobase (kb) Eco RI DNA fragment from normal mouse Balb/c genomic DNA that contains sequences (sarc) homologous to the acquired cell sequences (src) of Moloney sarcoma virus (MSV) has been cloned in phage lambda. The sarc region (1.2 to 1.3 kb) of the 15.0-kb cell fragment is indistinguishable from the src region of two isolates of MSV as judged by heteroduplex and restriction endonuclease analyses. The cellular sequences flanking sarc show no homology to other MSV sequences. Whereas cloned subgenomic portions of MSV that contain src transformed NIH-3T3 cells in vitro, the cloned sarc fragment is inactive.     

小鼠FGF9在大肠杆菌中的表达与纯化

为了建立小鼠FGF9的原核表达体系,获得纯化的FGF9重组蛋白,采用PCR技术扩增FGF9,并将其克隆入原核表达载体pET30a(+)中,经测序验证后将该重组质粒转入大肠杆菌BL21(DE3)中进行表达,采用亲和层析技术进行目的蛋白的纯化,并用Western blotting进行了免疫原性验证.测序结果显示插入到载体中的片段与FGF9的天然序列一致,在BL21(DE3)中表达出了可溶性的FGF9重组蛋白,其表达量达到总蛋白的40 %;用镍螯合亲和层析方法获得了纯度大于95 %的FGF9重组蛋白.     

Pressure-induced phases of sulfur

At least three phases of sulfur may be induced at pressures between 16 and 65 kilobars. A fibrous form obtained at pressures of 27 kilobars and higher appears to be closely related to the form obtained when plastic sulfur is chilled and stretched. Crystals of the fibrous form are orthorhombic with a = 13.8, b = 32.4, and c = 9.25 A. Thus far the results are in accord with deductions made by Prins and co-workers that the sulfur atoms are arranged in helices with ten atoms and three turns per 13.8-A period, and that these helices are essentially close-packed. The unit cell contains 16 ten-atom chain lengths. The probable space groups to which the crystal may belong are C c m m, C c 2 m, or C c m 2(1). These imply that the structure must contain both right- and left-handed helices and that at least half the helices have some disorder about their axes. The other two phases appear to have structures related to that of the fibrous form, but analyses of them has not progressed as far. One of these phases appears to be uomegasulfur.     

The amyloid beta protein gene is not duplicated in brains from patients with Alzheimer's disease

Complementary DNAs (cDNAs) encoding portions of the amyloid beta protein were used to investigate possible amyloid gene duplication in sporadic Alzheimer's disease. A strategy employing two Eco RI restriction fragment length polymorphisms (RFLPs) detected by the amyloid cDNAs was used. RFLPs allow the detection of a 2:1 gene dosage in the DNA of any individual who is heterozygous for a particular RFLP. The amyloid gene regions homologous to the cDNAs used were not duplicated in the DNA from brains of individuals with sporadic Alzheimer's disease. Similar results were also obtained with a strategy employing a test for 3:2 gene dosage.     

遗传工程稻叶绿体psbA基因的克隆及结构分析

分别制备了遗传工程稻ＧＥＲ－１及其ＤＮＡ供体（慈利玉米）、ＤＮＡ受体（水稻湘早籼８号）的叶绿体ＤＮＡ（ｃｐＤＮＡ），对三者之间的多种限制性内切酶酶切图谱比较表明，ＧＥＲ－１的ｃｐＤＮＡ基本与ＤＮＡ受体水稻一致．进一步克隆了ＣＥＲ－１的ｐｓｂＡ基因，并构建其酶切图谱、测定其３′非翻译区（３′ＵＴＲ）序列，初步认为该基因的序列结构与水稻相同，从而排除了ＧＥＲ－１的性状变异是由其ｐｓｂＡ基因发生变化所致的可能．此外，对叶绿体ＤＮＡ的制备及酶切消化作了较深入的探讨     

牛eIF5基因原核表达与蛋白纯化

研究应用PCR方法扩增牛e IF5基因编码序列,通过Eco RⅠ和SalⅠ酶切位点将其定向插入p GEX-4T-1载体,构建原核表达重组质粒p GEX-4T-1-e IF5,并转化大肠埃希菌BL21。酶切和测序鉴定正确后,用异丙基硫代-β-D-半乳糖苷(IPTG)大量诱导表达,利用GST-Sepharose 4B亲和珠纯化,SDS-PAGE和Western Blot鉴定。结果表明,原核表达载体p GEX-4T-1-e IF5构建成功,Western Blot证实融合蛋白大量表达,纯化后获得GSTe IF5融合蛋白,为深入研究牛e IF5蛋白功能奠定基础。     

A human Y-linked DNA polymorphism and its potential for estimating genetic and evolutionary distance

A human DNA sequence (p12f2), derived from a partial Y-chromosome genomic library and showing homology with the X and Y chromosomes and with an undetermined number of autosomes, detected two Y-specific restriction fragment length variants on male DNA that had been digested with Taq I and Eco RI. These variants may have been generated through a deletion-insertion mechanism and their pattern of holoandric transmission indicates that they represent a two-allele Y-linked polymorphism (RFLP). By means of DNA from patients with inborn deletions in chromosome Y, this polymorphic DNA site was mapped to the interval Yq11.1-Yq11.22. The frequency of the rarest allele was about 35 percent in Algerian and Sardinian human males, whereas it was only 4 percent among Northern Europeans. The p12f2 probe also detected Y-specific DNA fragments in the gorilla and chimpanzee. In view of the monosomy of the Y chromosome in mammalian species, Y-linked RFLP's may prove to be more useful than autosomal or X-linked markers in estimating genetic distances within and between species.     

Fragile X genotype characterized by an unstable region of DNA

DNA sequences have been located at the fragile X site by in situ hybridization and by the mapping of breakpoints in two somatic cell hybrids that were constructed to break at the fragile site. These hybrids were found to have breakpoints in a common 5-kilobase Eco RI restriction fragment. When this fragment was used as a probe on the chromosomal DNA of normal and fragile X genotype individuals, alterations in the mobility of the sequences detected by the probe were found only in fragile X genotype DNA. These sequences were of an increased size in all fragile X individuals and varied within families, indicating that the region was unstable. This probe provides a means with which to analyze fragile X pedigrees and is a diagnostic reagent for the fragile X genotype.     

Microtubule-macrotubule transitions: intermediates after exposure to the mitotic inhibitor vinblastine

Vinblastine treatment of microtubule protein or intact microtubules assembled in vitro produced bifilar rings and bifilar helices. Suspentsions of rings and helices were demonstrated to bind [(3)H]colchicine, a diagnostic property of microtubule protein. Macrotubules are suggested to consist of tightly coiled helices formed by longitudinal compacting of loosely coiled protofilament pair intermediates.     

血管内皮生长因子（VEGF）实时荧光定量PCR质粒标准品的构建

 应用SYBR Green荧光染料检测方法构建VEGF基因质粒标准品,能快速、灵敏、可靠的检测与临床多种疾病密切相关的重要候选基因血管内皮生长因子（VEGF）的表达情况。本研究利用版纳微型猪近交系4~6月龄猪,提取皮肤创面总RNA,设计特异引物,进行RT PCR扩增。纯化目的片段并与pMD18 T载体连接,转化宿主菌DH5α,提取重组质粒DNA,并经酶切、PCR和测序鉴定,计算重组质粒原液拷贝数浓度并制备梯度浓度标准品,进行实时荧光定量PCR。建立的VEGF基因mRNA表达实时荧光定量PCR检测方法特异性较好,检测灵敏度可达1.0×103拷贝,线性范围达1.0×103~1.0×108拷贝,阈值循环数(Ct)与PCR体系中起始模板量的对数值之间存在良好的线性关系(r2=0.982),扩增效率高(E=96.92％)。通过构建版纳微型猪近交系VEGF基因质粒标准品,为探讨VEGF基因在临床多种疾病中所发挥的分子机理奠定基础。     

DNA binding by proteins

Study of proteins that recognize specific DNA sequences has yielded much information, but the field is still in its infancy. Already two major structural motifs have been discovered, the helix-turn-helix and zinc finger, and numerous examples of DNA-binding proteins containing either of them are known. The restriction enzyme Eco RI uses yet a different motif. Additional motifs are likely to be found as well. There is a growing understanding of some of the physical chemistry involved in protein-DNA binding, but much remains to be learned before it becomes possible to engineer a protein that binds to a specific DNA sequence.