遗传学实验技术

Single-strand conformational polymorphism (SSCP) analysis is a method for screening for mutations. This method is based on observations of Orita and coworkers that single strands of DNA assume unique conformations depending on their primary sequence. The uniqueness of each three- ...

DNA gyrase catalyzes DNA supercoiling in a reaction coupled to ATP hydrolysis (1). The enzyme has been found in many eubacterial species and is unique among the topoisomerases in promoting negative supercoiling of DNA (2,3). A variety of studies have shown that gyrase is essential for bacterial ...

A key aspect of anti-topotsomerase drug action is that most antitopoisomerase drugs act by stabilizing an intermediate of the topoisomerase reaction (1). This intermediate consists of the enzyme covalently bound to DNA by a phosphotyrosine linkage, where the DNA strand scission has occ ...

A major role of DNA topoisomerase II in vivo is to catalyze the double-stranded cleavage of DNA, allowing passage of a second DNA duplex through the break. This activity requires adenosine triphosphate (ATP) and is necessary for separating catenated DNA duplexes found at the end of replication. ...

In “inventing” reverse gyrase, the living world has built a unique and remarkably sophisticated enzyme that is more than a simple topoisomerase. Reverse gyrase possesses the unique ability to catalyze the production of positive supercoils in a closedcircular DNA at the expense of ATP (1); see ...

A collection of chapters assembled in this volume provides an illustration of remarkable technological progress in genome-scale essentiality analysis in a variety of microbial species. In accord with other genomic techniques, one may anticipate that the volume of essentiality da ...

In this chapter, the process for the reconstruction of genome-scale metabolic networks is described, and some of the main applications of such models are illustrated. The reconstruction process can be viewed as an iterative process where information obtained from several sources is com ...

Although the emergence and spread of antimicrobial resistance in major bacterial pathogens for the past decades poses a growing challenge to public health, discovery of novel antimicrobial agents from natural products or modification of existing antibiotics cannot circumvent ...

Essential genes are the genes that are indispensable for the survival of an organism. The genome-scale identification of essential genes has been performed in various organisms, and we consequently constructed DEG, a Database that contains currently available essential genes. Here ...

The Profiling of Escherichia coli Chromosome (PEC) database (http://www.shigen.nig.ac.jp/ecoli/pec/) is designed to allow E. coli researchers to efficiently access information from functional genomics studies. The database contains two principal types of data: gene essenti ...

TAG, or bar-code, microarrays allow measurement of the oligonucleotide sequences (TAGs) that mark each strain of deletion mutants in the Saccharomyces cerevisiae yeast knockout (YKO) collection. Comparison of genomic DNA from pooled YKO samples allows estimation of relative abun ...

Mutant propagation (outgrowth) is an important step in all large-scale gene essentiality experiments, profoundly influencing essentiality assignment produced. Using a simplified mathematical model of competitive outgrowth in a diverse mutant population, we have identi ...

As transposomics is extended to genome scale, appropriate statistical methods need to be developed to assign significance to gene essentiality. In this chapter, the author presents a set of steps that, together with genome-scale insertion data and the complete genome sequence of a prokar ...

During the construction of random transposon mutagenesis libraries, four essential statistical issues arise: (1) Computing basic probability results for number of open reading frame knockouts. (2) Estimating the number of new open reading frames that will be knockouts in the next set of ...

We have constructed a near-saturation level mutant library for Pseudomonas aeruginosa strain PAO1 using Tn5-derived transposons mapped to the PAO1 reference sequence. This chapter describes the high-throughput techniques used to generate and map the mutant strains. In addition, ...

Transposons have long been recognized as useful laboratory tools facilitating genome-scale studies of gene function. Relative to traditional methods, transposon mutagenesis offers a rapid and economical means of generating large numbers of independent insertions in target ...

Staphylococcus aureus is the leading cause of wound and hospital-acquired infections. The emergence of strains with resistance to all antibiotics has created a serious public health problem. Transposon-based mutagenesis can be used to generate libraries of mutants and to query geno ...

We present a whole-genome approach to genetic footprinting in Escherichia coli using Tn5-based transposons to determine gene essentiality. A population of cells is mutagenized and subjected to outgrowth under selective conditions. Transposon insertions in the surviving muta ...

PCR-based signature tagged mutagenesis is an “en masse” screening technique based upon unique oligonucleotide tags (molecular barcodes) for identification of genes that will diminish or enhance maintenance of an organism in a specific ecological niche or environment. PCR-based ...

Microarray mapping of transposon insertions can be used to quantify the relative abundance of different transposon mutants within a complex pool after exposure to selective pressure. The transposon site hybridization (TraSH) method applies this strategy to the study of Mycobacte ...