实验基础

Borrelia burgdorferi is an etiologic agent of Lyme disease, the most common arthropod-borne disease in the United States (1,2). The bacterium, a member of the spirochete phylum, has a genome predominantly composed of linear DNA molecules (3,4). Formulating a medium in which B. burgdorferi gro ...

Transformation methods for the baker’s yeast Saccharomyces cerevisiae have allowed for rapid advances in molecular genetic studies with this model microorganism, for the development of alternative expression systems to prepare recombinant proteins, and for the development ...

Many of the techniques that have been developed for the manipulation of the budding yeast Saccharomyces cerevisiae have now been adapted to be used on the alternative host, Schizosaccharomyces pombe. One particularly important technique is the introduction of exogenous DNA into the ye ...

The introduction of exogenous DNA into prokaryotic and eukaryotic cells is one of the most frequently used procedures in the daily laboratory work of a molecular geneticist. In recent years, methods based on high-voltage electric shocks have been established for various species (revie ...

The fungi encompass an enormous array of species, ranging from microscopic uninucleate, unicellular forms to multinucleate, coenocytic, highly differentiated macroscopic morphological forms. The wealth of diversity provided by a wide variety of life cycles, the availability ...

The genus Candida comprises a group of yeasts united by nothing more than the fact that none has a natural sexual cycle. If these yeasts do not form a natural grouping, they nonetheless contain a number of species of considerable scientific and practical interest. Candida albicans is an important o ...

The protist Physarum polycephalum is a convenient system for studies of molecular and cellular biology of fundamental eukaryotic processes, including DNA replication, mitotic regulation, single-cell development, the cytoskeleton, and motility. The life cycle of this acellu ...

Dictyostelium discoideum has long been an intriguing model system for the study of cell type divergence during development, cell signaling, gene expression, and other cell biological problems (1). With the development of DNA-mediated transformation, research in Dictyostelium h ...

Tetrahymena thermophila is a free-living ciliated protozoan useful in cell biological and molecular genetic studies. Recently DNA-mediated transformation has been developed for this microorganism (1), and used to study the mechanisms of genome rearrangement (2–4) and structu ...

Electroporation was first applied in Giardia lamblia in 1990 by Furfine and Wang (1) to introduce a full-length giardiavirus (GLV) single-stranded RNA (ssRNA) into uninfected G. lamblia cells. After electroporation, the cells were grown to confluency and passed serially at 1∶13 dilutio ...

The protozoan genus Trypanosoma contains several pathogenic parasites of major medical and veterinary importance. These include Trypanosoma brucei subspecies, the causative agents of African trypanosomiasis, and Trypanosoma cruzi, the causative agent of American trypa ...

A common limitation in any molecular biological study is the introduction of DNA, whether bacteriophage or plasmid, into a recipient strain. Electroporation has a number of advantages over other more traditional methods for the introduction of DNA into cells, the most significant being t ...

In the late 1970s and early 1980s, several laboratories (1–4) reported that an applied electric field could induce transient pores in eukaryotic cell membranes. In addition to small molecules, such as sucrose and dyes, DNA could be introduced into these electropermeabilized cells (5–7). Mo ...

The cytoplasmic regulation of gene expression has received increased attention in recent years. In order to assess directly the in vivo impact of regulatory elements on the translational efficiency and stability of an mRNA in higher eukaryotes, a variety of methods that deliver RNA direct ...

Electrofusion is an important method in genetics and biotechnology. The first morphological (1) and genetical (2) evidence for yeast electrofusion was presented at the 5th Bioelectrochemical Symposium held at Weimar in 1979. Protoplasts from auxotrophic yeast strains were fused in a ...

Electroporation is now being used to transfer into cells a variety of macromolecules, including DNA, RNA, protein, fluorescent dyes, and some chemotherapeutic agents. With electrotransformation, as many as 80% of the cells receive exogenous DNA (1). Chemical and natural methods of DNA tra ...

Enteric bacteria are not naturally competent for DNA-mediated transformation, but methods derived from the Ca2+-shock method of Mandel and Higa (1) permit uptake of DNA at levels adequate for molecular cloning. Transformation frequencies using Ca2+-shock methods with selected str ...

Fusion mediated by the human immunodeficiency virus type-1 (HIV-1) envelope (Env) glycoprotein and the cellular CD4/chemokine receptor complex is the first step in entry and is often analyzed in cell-cell fusion assays that require Env expression by recombinant vaccinia viruses and/ ...

Poxvirus DNA is not infectious because the initiation of the infective process requires proteins encapsidated along with the virus genome. However, infectious virus can be produced if purified poxvirus DNA is transfected into cells previously infected with another poxvirus. This p ...

In the procedures described here, respiratory metabolism is estimated by direct determination of oxygen uptake, measured as a reduction in the dissolved oxygen tension (DOT) of cell suspensions or extracts. Continuous monitoring of DOT is achieved using a Clark-type oxygen electrode ...