微生物学技术

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 ...

Helicobacter pylori has been widely studied since its discovery in 1982 by Marshall and Warren (1), but many aspects of its structure, metabolism, and physiology, including its specific growth requirements, are still largely unknown, The organism is generally grown in complex media cont ...

Until recently, the culture of Helicobacter pylori in vitro has only been achievable with complex undefined media supplemented with blood products, such as Brain-heart infusion (BHI) medium, blood agar, chocolate agar, or lysed blood agar (1). Substrate utilization and metabolic acti ...

The human mitochondrial genome is composed of a 16-kb circular, doublestranded DNA that encodes 13 polypeptides of the mitochondrial respiratory chain, 22 transfer RNAs, and 2 ribosomal RNAs required for protein synthesis (1). It is generally accepted that mitochondrial DNA (mtDNA) mu ...

We have developed an in situ polymerase chain reaction (PCR) assay for the elucidation of the human mitochondrial DNA4977 deletion (common mtDNA deletion), which occurs between two 13-bp direct repeats situated 4977 bp apart (1). Although the origin of the common mtDNA deletion is unknown, its ...

The budding yeast Saccharomyces cerevisiae is a particularly suitable organism for identifying nuclear genes involved in the maintenance of the mitochondrial genome. Indeed, S. cerevisiae can grow and divide in the absence of respiration and, moreover, without mitochondrial DNA. In ...

Recent developments in PCR fluorimetry have allowed for quick quantification of target molecules. Before the invention of fluorimetric quantitative PCR, researchers who wanted to quantify the amount of a gene in a sample did so painstakingly by limiting dilution, competitive polym ...

Mitochondrial mutation rates have historically been more refractory to genetic analysis than nuclear mutation rates. This is the result of the lack of auxotrophic genes in the mitochondria, the multiple (50-100) copies of mitochondrial genomes per cell (1), the random segregation of mit ...

Ever since the demonstration that mitochondria contain DNA (mtDNA) and a complete apparatus for its replication and expression there has been an interest in identifying the enzymes involved in these processes. This problem is compounded by the fact that similar DNA transactions also occ ...

Affinity chromatography is a powerful technique that allows purification of proteins based on biospecific interactions using an affinity absorbent to selectively bind a target protein (1,2). Separation is achieved through a highly specific, but reversible, interaction with a co ...

Mitochondria are cytoplasmic organelles that generate cellular energy in the form of ATP by the process of oxidative phosphorylation. Mitochondria contain multiple copies of a 16.5-kb circular genome. Mitochondrial DNA (mtDNA) encodes a subset of 13 structural, 22 transfer (tRNA), and 2 ...

The human mitochondrial genome encodes a variety of genes required for oxidative phosphorylation, and loss of these essential gene functions induces a multitude of severe metabolic disorders (1–4). Mitochondrial DNA (mtDNA) is replicated by the nuclear encoded DNA polymerase γ (5), and ...

Mitochondrial DNA (mtDNA) replication is a complex process involving over 20 proteins organized along the inner mitochondrial membrane as a multienzyme complex called the mtDNA replisome, or replication factory (1–3). Figure 1 illustrates some of the protein components that parti ...

The identification of proteins involved in mitochondrial biogenesis remains an important challenge. In the past decade, it has become clear that a biochemical approach is limited by the investigator’s ability to develop a highly specific assay that differentiates the mitochondri ...