微生物学技术

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 ...

Respiratory chain dysfunction is an important contributor to human pathology (1–3). The generation of animal models has much facilitated in-depth studies of pathogenetic mechanisms in mitochondrial disease (4). The function of the respiratory chain is subject to the dual genetic con ...

Mitochondrial DNA (mtDNA) nucleoids have been isolated from several organisms, including rat (liver) (1), Physarum polycephalum (2), Saccharomy- ces cerevisiae (3) and Pichia jadinii (4). Most methods for nucleoid isolation have utilized detergent extraction and sucrose gradie ...

The development of baculovirus expression vector systems has accompanied a rapid expansion of our knowledge about the genes, their function, and regulation in insect cells. Classification of these viruses has also been refined as we learn more about differences in gene content between i ...

The insect cell culture/baculovirus system has three primary applications: (1) recombinant protein synthesis, (2) biopesticide synthesis, and (3) as a model system (e.g., for studying apoptosis). The fundamental techniques involved in these applications are described througho ...

The production of a recombinant baculovirus expression vector normally involves mixing infectious virus DNA with a plasmid-based transfer vector and then cotransfecting insect cells to initiate virus infection. The aim of this chapter is to provide an update on the range of baculovirus ...

Although there are several different methods available of making recombinant baculovirus expression vectors (reviewed in Chapter 3), all require a stage in which insect cells are transfected with either the virus genome alone (Bac-to-Bac� or BaculoDirect™, Invitrogen) or virus geno ...

This chapter lists the known cell lines from Lepidoptera, largely based on previous compilations of insect cell lines published by W. Fred Hink. The official designation is given for each cell line as well as the species, tissue source, and, when known, the susceptibles to baculoviruses.

BacMams are modified baculoviruses that contain mammalian expression cassettes for gene delivery and expression in mammalian cells. The BacMam system combines the advantages of viral transient expression, ease in generation, and a wide cell tropism. It enables rapid, facile, and fle ...

Lepidopteran insect cell culture technology has progressed to the point of becoming an essential part of one of the most successful eukaryotic expression systems and is increasingly used industrially on a large scale. Therefore, there is a constant need for convenient and low-cost cultu ...

This chapter describes procedures for initiating new cell lines from lepidopteran larval tissues. The internal morphology is described along with methods for treating excised tissues and the primary cultures. Advice on culture medium and the tissues that will provide the best chance f ...

The various methods for maintaining (a.k.a., subculturing, splitting, or passaging) established lepidopteran cell lines are described. Three procedures are presented that are appropriate for different cell lines dependent upon the growth characteristics (in particular, ce ...

The production of recombinant proteins using the baculovirus expression vector system in large-scale agitated bioreactors is discussed in this chapter. Detailed methods of the key stages of a batch process, including host cell growth, virus stock amplification and quantificatio ...

Improved methods of baculovirus cloning and insect cell culture and their commercialization have made the use of the baculovirus expression vector system (BEVS) a routine tool for the production of preparative quantities of recombinant protein. This chapter outlines basic techni ...