Calicheamicin gamma 1I and DNA: molecular recognition process responsible for site-specificity

Calicheamicin gamma 1I is a recently discovered diyne-ene-containing antitumor antibiotic that cleaves DNA in a double-stranded fashion, a rarity among drugs, at specific sequences. It is proposed that the cutting specificity is due to a combination of the complementarity of the diyne-ene portion of the aglycone with DNA secondary structures and stabilization by association of the thiobenzoate-carbohydrate tail with the minor groove.     

Xeroderma pigmentosum group E cells lack a nuclear factor that binds to damaged DNA

The disease xeroderma pigmentosum is characterized by deficient repair of damaged DNA. Fusions of cells from different patients have defined nine genetic complementation groups (A through I), implying that DNA repair in humans involves multiple gene products. In this report, an extension of the gel electrophoresis binding assay was used to identify at least one nuclear factor that (i) bound to DNA damaged by ultraviolet radiation or the antitumor drug cisplatin, but (ii) was notably absent in cells from complementation group E. Therefore, the factor appears to participate in a versatile DNA repair pathway at the stage of binding and recognition.     

Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II

Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.     

Association of crossover points with topoisomerase I cleavage sites: a model for nonhomologous recombination

Nonhomologous DNA recombination is frequently observed in somatic cells upon the introduction of DNA into cells or in chromosomal events involving sequences already stably carried by the genome. In this report, the DNA sequences at the crossover points for excision of SV40 from chromosomes were shown to be associated with eukaryotic topoisomerase I cleavage sites in vitro. The precise location of the cleavage sites relative to the crossover points has suggested a general model for nonhomologous recombination mediated by topoisomerase I.     

Erythropoietin retards DNA breakdown and prevents programmed death in erythroid progenitor cells

The mechanism by which erythropoietin controls mammalian erythrocyte production is unknown. Labeling experiments in vitro with [3H]thymidine demonstrated DNA cleavage in erythroid progenitor cells that was accompanied by DNA repair and synthesis. Erythropoietin reduced DNA cleavage by a factor of 2.6. In the absence of erythropoietin, erythroid progenitor cells accumulated DNA cleavage fragments characteristic of those found in programmed cell death (apoptosis) by 2 to 4 hours and began dying by 16 hours. In the presence of erythropoietin, the progenitor cells survived and differentiated into reticulocytes. Thus, apoptosis is a major component of normal erythropoiesis, and erythropoietin controls erythrocyte production by retarding DNA breakdown and preventing apoptosis in erythroid progenitor cells.     

Reaction of the antitumor antibiotic CC-1065 with DNA: structure of a DNA adduct with DNA sequence specificity

Sequence-dependent variations in DNA revealed by x-ray crystallographic studies have suggested that certain DNA-reactive drugs may react preferentially with defined sequences in DNA. Drugs that wind around the helix and reside within one of the grooves of DNA have perhaps the greatest chance of recognizing sequence-dependent features of DNA. The antitumor antibiotic CC-1065 covalently binds through N-3 of adenine and resides within the minor groove of DNA. This drug overlaps with five base pairs for which a high sequence specificity exists.     

Structural basis of type II topoisomerase inhibition by the anticancer drug etoposide

Type II topoisomerases (TOP2s) resolve the topological problems of DNA by transiently cleaving both strands of a DNA duplex to form a cleavage complex through which another DNA segment can be transported. Several widely prescribed anticancer drugs increase the population of TOP2 cleavage complex, which leads to TOP2-mediated chromosome DNA breakage and death of cancer cells. We present the crystal structure of a large fragment of human TOP2β complexed to DNA and to the anticancer drug etoposide to reveal structural details of drug-induced stabilization of a cleavage complex. The interplay between the protein, the DNA, and the drug explains the structure-activity relations of etoposide derivatives and the molecular basis of drug-resistant mutations. The analysis of protein-drug interactions provides information applicable for developing an isoform-specific TOP2-targeting strategy.     

Cleaving DNA at any predetermined site with adapter-primers and class-IIS restriction enzymes

A four-component system has been designed that makes it possible to prepare a double-stranded (ds) DNA fragment; one fragment end is predesigned (by the use of a class-IIS restriction enzyme and adapter-primer), and the other end corresponds to any normal restriction cut. The system is composed of the phage M13mp7 single-stranded (ss) target DNA; the Fok I restriction enzyme; an oligodeoxynucleotide adapter-primer, which permits one to introduce Fok I cuts at any specified site in the target DNA; and DNA polymerase, which converts the ss target into a ds form ready for cloning. In this system, the oligodeoxynucleotide adapter-primer serves several purposes. The 5' hairpin ds domain of the adapter-primer contains the Fok I recognition site. Its 3' ss domain selects a complementary site on the target ss DNA, hybridizes with it to form the ds cleavage site, and serves as a primer to convert the ss M13mp7 target to ds DNA.     

Three-dimensional structure of the Tn5 synaptic complex transposition intermediate

Genomic evolution has been profoundly influenced by DNA transposition, a process whereby defined DNA segments move freely about the genome. Transposition is mediated by transposases, and similar events are catalyzed by retroviral integrases such as human immunodeficiency virus-1 (HIV-1) integrase. Understanding how these proteins interact with DNA is central to understanding the molecular basis of transposition. We report the three-dimensional structure of prokaryotic Tn5 transposase complexed with Tn5 transposon end DNA determined to 2.3 angstrom resolution. The molecular assembly is dimeric, where each double-stranded DNA molecule is bound by both protein subunits, orienting the transposon ends into the active sites. This structure provides a molecular framework for understanding many aspects of transposition, including the binding of transposon end DNA by one subunit and cleavage by a second, cleavage of two strands of DNA by a single active site via a hairpin intermediate, and strand transfer into target DNA.     

Uracil DNA glycosylase activity is dispensable for immunoglobulin class switch

Activation-induced cytidine deaminase (AID) is required for the DNA cleavage step in immunoglobulin class switch recombination (CSR). AID is proposed to deaminate cytosine to generate uracil (U) in either mRNA or DNA. In the second instance, DNA cleavage depends on uracil DNA glycosylase (UNG) for removal of U. Using phosphorylated histone gamma-H2AX focus formation as a marker of DNA cleavage, we found that the UNG inhibitor Ugi did not inhibit DNA cleavage in immunoglobulin heavy chain (IgH) locus during CSR, even though Ugi blocked UNG binding to DNA and strongly inhibited CSR. Strikingly, UNG mutants that had lost the capability of removing U rescued CSR in UNG-/- B cells. These results indicate that UNG is involved in the repair step of CSR yet by an unknown mechanism. The dispensability of U removal in the DNA cleavage step of CSR requires a reconsideration of the model of DNA deamination by AID.     

Genetic control of two gamma-globulin isoantigenic sites in domestic rabbits

Results of immunochemical analysis of sera from 335 offspring of 81 litters of rabbits are consistent with the hypothesis that the isoantigenic sites, RGG-I and RGG-II, of the gamma-globulins are controlled by a single allelic pair of autosomal genes with both specificities exhibited by the heterozygote. The three genotypes may be designated gamma(I)/gamma(I), gamma(II)/gamma(II), and gamma(I)/gamma(II).     

Conferring operator specificity on restriction endonucleases

Mapping and manipulation of very large genomes, including the human genome, would be facilitated by the availability of a DNA cleavage method with very high site specificity. Therefore, a general method was devised that extends the effective recognition sequences well beyond the present 8-base pair limit by combining the specificity of the restriction endonuclease with that of another sequence-specific protein that binds tightly to DNA. It was shown that the tightly binding lac or lambda repressor protects a restriction site within the operator from specific modification methylases, M.Hha I or M.Hph I, while all other similar sites are methylated and thus rendered uncleavable. A plasmid containing a symmetric lac operator was specifically cleaved by Hha I, only at the site within the operator, after M.Hha I methylation in the presence of the lac repressor, whereas the remaining 31 Hha I sites on this plasmid were methylated and thus not cleaved. Analogous results were obtained with the Hae II site within the lac operator, which was similarly protected by the lac repressor, and with the Hph I site within the phage lambda oL operator, which was protected by lambda repressor from M.Hph I methylation.     

猪生殖和呼吸综合征中国分离株ORF5 基因的克隆及序列分析

本研究利用对应于PRRSV ATCC VR-2332株及LV株ORF     

Immunochemical proof that a novel rearranging gene encodes the T cell receptor delta subunit

The T cell receptor (TCR) delta protein is expressed as part of a heterodimer with TCR gamma, in association with the CD3 polypeptides on a subset of functional peripheral blood T lymphocytes, thymocytes, and certain leukemic T cell lines. A monoclonal antibody directed against TCR delta was produced that binds specifically to the surface of several TCR gamma delta cell lines and immunoprecipitates the TCR gamma delta as a heterodimer from Triton X-100 detergent lysates and also immunoprecipitates the TCR delta subunit alone after chain separation. A candidate human TCR delta complementary DNA clone (IDP2 O-240/38), reported in a companion paper, was isolated by the subtractive library approach from a TCR gamma delta cell line. This complementary DNA clone was used to direct the synthesis of a polypeptide that is specifically recognized by the monoclonal antibody to TCR delta. This complementary DNA clone thus corresponds to the gene that encodes the TCR delta subunit.     

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.     

Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes

Single base substitutions can be detected and localized by a simple and rapid method that involves ribonuclease cleavage of single base mismatches in RNA:DNA heteroduplexes. A 32P-labeled RNA probe complementary to wild-type DNA is synthesized in vitro and annealed to a test DNA containing a single base substitution. The resulting single base mismatch is cleaved by ribonuclease A, and the location of the mismatch is then determined by analyzing the sizes of the cleavage products by gel electrophoresis. Analysis of every type of mismatch in many different sequence contexts indicates that more than 50 percent of all single base substitutions can be detected. The feasibility of this method for localizing base substitutions directly in genomic DNA samples is demonstrated by the detection of single base mutations in DNA obtained from individuals with beta-thalassemia, a genetic disorder in beta-globin gene expression.     

芝芪菌质多糖的分离、纯化及体外抗肿瘤活性初步研究

采用水提、醇沉、离子交换层析等方法分离纯化芝芪菌质多糖,并对各纯化多糖成分进行体外肿瘤细胞增殖的抑制活性测定。用DEAE-32纤维素柱层析分离得到4种纯化的芝芪菌质多糖GAP-1、GAP-2、GAP-3、GAP-4。4种纯化的芝芪菌质多糖均有一定程度的体外抗肿瘤活性,其中适宜浓度的GAP-3的体外肿瘤细胞增殖的抑制活性最好。芝芪菌质纯化多糖对GAP-3K562、SMMC-7721和PC-3细胞增殖抑制作用明显,有一定的抗肿瘤药开发前景。     

猪线粒体DNA D-loop PCR-RFLP分析

对太湖猪（二花脸）、大约克猪、北京黑猪、五指山猪、香猪、长白猪、杜洛克猪以及太湖猪与7个品种猪的正反交杂交家系线粒体DNAD－loopPCR－RFLP分析,发现猪的线粒体DNAD－loop约为1500bp,长白猪及部分大约克猪与其他猪品种在AccⅠ酶切位点存在多态性;太湖猪、大约克猪的正反交后代的线粒体DNAD－loop在AccⅠ酶切位点存在多态性,其遗传方式遵循母系遗传特性。