实验基础

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 ...

Single-stranded DNA-binding proteins (SSBs) serve critical roles in DNA replication, repair, and recombination (1). Mitochondrial SSBs (mtSSBs) share similar physical and biochemical properties with Escherichia coli SSB (2–7), with which they exhibit a high degree of amino acid se ...

At least two DNA helicases are important for maintenance of the yeast Saccharomyces cerevisiae mitochondrial DNA (mtDNA). Pif1 protein is a 5′-3′ DNA helicase that affects the stability of mtDNA at higher temperatures but is not essential at 28�C. Pif1 is probably involved in mtDNA repair and rec ...

The mitochondrial single-stranded DNA-binding protein (mtSSB) is encoded by a single-copy nuclear gene in mammals. After de novo synthesis in the cytosol, mtSSB is transported into mitochondria, where it participates in mitochondrial DNA (mtDNA) replication, RNA transcription, a ...

DNA topoisomerases constitute a ubiquitous class of enzymes that engage chromosomal DNA and promote the passage of the two strands of the DNA helix through one another (reviewed in refs. 1 and 2). This occurs while preserving the phosphodiester backbone of each strand. In some instances, the DNA t ...

Mitochondria in mammalian cells contain a nuclearly encoded, Mg+2 (Mn+2)-dependent DNA endonuclease activity (reviewed in ref. 1), that has been termed Endonuclease G (or Endo G) (2). This enzyme activity, an approx 60-kDa homodimer of a 29-kDa polypeptide, accounts for nearly all of the potent D ...

Studies of molecular mechanisms coordinating mammalian mitochondrial replicative and transcriptional processes have, for the most part, been limited to in vitro analyses. Although much has been learned from in vitro studies (1), they are often difficult to develop and may not depict bi ...

Mitochondrial DNA is a very vulnerable target for both alkylating and oxidizing agents (1–5). Recent studies from our laboratory demonstrated an association between inefficient mtDNA repair capacity and an increased sensitivity to both types of DNA damaging agent (5–9). To determine if a ...