遗传学实验技术

The helical structure of duplex DNA allows for the faithful duplication and transmission of genetic information from one generation to the next, at the same time maintaining the integrity of the polynucleotide chains. The complementary nature of the two antiparallel DNA strands enables ...

The mechanism of action of DNA topoisomerase I in catalyzing the relaxation of supercoiled DNA and how this reaction is perturbed by the antitumor drug camptothecin have been the subject of intense investigation (reviewedin (1-3). Much of this effort has focused on structure/function stu ...

Mechanistic and structural studies require large quantities of highly purified enzyme. Unfortunately, traditional expression strategies using Escherichia coli are often not successful for eukaryotic proteins, especially large ones. Despite numerous attempts, overe ...

To characterize properly protein function and enzymatic activity, it is highly desirable to perform experiments with purified protein preparations. This is especially true in the case of topoisomerase II, because many of the enzymatic assays critical to the topoisomerase field (su ...

DNA topoisomerase I (topo I) has been firmly established as the molecular target of the camptothecin group of anticancer drugs. These drugs include camptothecin, topotecan, 9-amino-camptothecin, and irinotecan (1). Many of them are now in clinical trials and are showing activity against a ...

DNA topoisomerase I (topo I) can be isolated from cultured human cells in quantities that are more than sufficient for investigations into the ability of topo I to relax supercoiled DNA (250 μg/109 cells) (1,2). However, the production of human topo I (htopo I) in this manner becomes both costly and labor ...

Protein phosphorylation is almost certainly the most important posttranslational mechanism of enzyme regulation in eukaryotic cells (reviewed in 1). The equilibrium between phosphorylation by protein kinases and dephosphorylation by protein phosphatases modulates t ...

Eukaryotic DNA Topoisomerase II (Topo II) is an essential enzyme that catalyzes the relaxation of supercoiled DNA and the segregation of newly replicated chromosomes (1-3). The enzyme is highly conserved through evolution, and appears to result from the fusion of the A- and B-subunits of bact ...

Western blotting has been widely utilized to detect various polypeptides or polypeptide epitopes (e.g., posttranslational modifications) within cells (reviewed in 1-4). If the signals in samples being analyzed are compared to a suitable standard curve and appropriate internal st ...

DNA topoisomerases can be grouped in to three families based on biochemical properties and amino acid sequence. Following are multiple protein sequence alignments of the members of each of these families: the eukaryotic DNA topoisomerase I type, the DNA topoisomerase II type, and the DNA top ...

Gel electrophoresis in one dimension is often insufficient to distinguish various molecular species of DNA, since different conformers, and sometimes DNA molecules of totally different structures may have the same electrophoretic mobility. These DNA species can be resolved by two ...

Agarose-gel electrophoresis is used to separate DNA molecules on the basis of size and shape (1-4). Since DNA is negatively charged, the charge-to-mass ratio is constant. Thus, migration through agarose is inversely proportional to the size of the molecule. However, the electrophoretic mo ...

The topology of DNA affects a number of major biological processes (1-4). For example, processive enzymes, such as the RNA and DNA polymerases, have the capability of generating both positive and negative DNA supercoils during the process of transcription and replication, respectively. T ...

Palindromic DNA sequences have the potential to form branched structures called cruciforms, in which the interstrand base pairs within the symmetric region are replaced with intrastrand base pairs. Cruciforms can sometimes form in vivo (1), and circumstantial evidence suggests th ...

The kinetoplast DNA (kDNA) network of the protozoan parasite Crithidia fasciculata is a naturally occurring gigantic catenane containing several thousand DNA minicircles. Because of its unusual structure, kDNA is an excellent substrate to use in decatenation assays of topoisom ...

The structural organization of both prokaryotic and eukaryotic chromosomes has evolved following a common principle: the need for storing the genetic information in topologically independent domains consisting of one or more genes and of all the elements required in cis for their fun ...

Type II topoisomerases readily remove knots from DNA (1). Removal of knots can be assayed by gel electrophoresis (1), and this provides a convenient assay for type II topoisomerase activity, even in crude extracts. Such assays have been useful in screening drugs that are suspected to inhibit type II ...

Supercoiling, knotting, and catenation are three common higher-order structures involving coiling of the axis of double-stranded DNA. These forms appear as a result of a number of important biological activities, including topoisomerase action, DNA replication, and genetic reco ...

Suicide gene therapy provides a mechanism for sensitizing cells to normally non-toxic compounds or prodrugs. Genes are introduced into cancer cells thereby rendering them sensitive to prodrugs by virtue of the activity of the suicide gene product toward the prodrugs. The most widely inv ...

Most enzymes used for antibody-directed enzyme-prodrug therapy (ADEPT) and gene-directed enzyme-prodrug therapy (GDEPT) are of bacterial, yeast, or viral origin. These xenogeneic proteins can induce an immune response, which might be a hindrance to their application of these enzym ...