遗传学实验技术

Quidquid agis, prudenter agas et respice finem!—Whatever you do, do it wisely and consider the goal. In consideration of that sage advice, the chicken B-cell line DT40 is an excellent model cell system to study the function of vertebrate genes. In addition to being highly amenable to gene manipulati ...

To perform structure/function analyses of a protein in vivo, ideally one should be able to simultaneously abolish expression of the endogenous wild-type protein, substitute it with a form of the protein containing a targeted mutation, and analyze the functional consequences. Until rec ...

RNA interference (RNAi) is an evolutionarily conserved mechanism of posttranscriptional gene silencing induced by introducing the double-stranded RNAs (dsRNAs) into cells. Recent progress in RNAi-based gene-silencing techniques has revolutionarily advanced in studi ...

The yeast two-hybrid system is a useful tool for identifying new protein-protein interactions, and for the dissection of previously identified interactions. An important issue in protein-interaction studies is frequently that of determining whether a protein associates spec ...

Yeast two-hybrid systems are artificial genetic systems that allow identification and characterization of protein-protein interactions. One common limit to the use of these techniques is when the intrinsic property of “bait” proteins of interest transcriptionally autoacti ...

Synthetic Cys2His2 zinc finger domains with novel DNA-binding specificities can be identified from large randomized libraries using selection methodologies such as phage display. It has been previously demonstrated that a bacterial cell-based two-hybrid system is at least as ef ...

The cloning of megabase-size DNA fragments in yeast artificial chromosomes (YACs) creates new opportunities to study, for example, remote cis-acting elements, higher order chromatin structure, and chromosomal folding in coordinating the transcriptional regulation of large ...

Mitotic crossing over was first observed in the somatic tissues of genetically marked strains of fruitfly (1), but has proved most valuable as a mapping tool in asexual fungi (2,3). Although the yeast Saccharomyces cerevisiae has a sexual cycle, and is therefore more amenable to genetic analysis ...

The ability to propagate long fragments of DNA cloned in Saccharomyces cerevisiae as yeast artificial chromosomes (YACs) represents a significant advance in cloning technology. Its introduction in 1987 (1) demonstrated the cloning of human DNA with the pYAC series of vectors in the yeast ...

Increasingly, the libraries that are the basic genomic DNA resources of physical mapping projects are stored in ordered arrays in the wells of 96- or 384-well microtiter plates (see Chapters 2–4). Localization of individual genomic clones to single wells of 96-well microtiter plates rather ...

Yeast artificial chromosome (YAC) clones are propagated in yeast, a host organism with a variety of established techniques for altering DNA sequences by homologous recombination in vivo. The modification of existing YAC clones allows the removal of undesired insert DNA (e.g., neighbor ...

The large regions of DNA that can be cloned in yeast artificial chromosomes (YACs) are ideal for expression studies of the complex genes and gene clusters found in the mammalian genome. Such studies require that the YAC of interest be transferred into a suitable expression system, such as mammali ...

Soon after the first report of how yeast artificial chromosomes (YACs) could be used as cloning vectors for large DNA fragments, the transfer of YACs into mammalian cells came into focus of interest, Following mammalian cell transfer, the YAC integrates into the host genome. Because of the large s ...

The isolation of genes from large candidate regions is one of the major problems for the molecular biologist. With the advent of yeast artificial chromosomes (YACs), the problem of cloning these regions is now largely solved; however, screening these large genomic regions for expressed seq ...

The use of baker’s yeast, Saccharomyces cerevisiae, for the cloning of extremely large genomic intervals (exceeding 1 Mb) was made possible with the development of yeast artificial chromosomes (YACs) (1). YACs are linear molecules containing all the control elements necessary for stab ...

One of the major efforts in the field of positional cloning and the human genome project is to identify coding sequences or transcription units in large genomic regions such as yeast artificial chromosomes (YACs). The task proves to be challenging because only a small percent of the total DNA of the ge ...

The genetic markers used in yeast, and in yeast artificial chromosome (YAC) cloning, are largely defined by the manipulation of growth media. This chapter, apart from providing recipes for media formulation, also contains a brief introduction to genetic markers in yeast that are relevant to Y ...

The storage of yeast artificial chromosome (YAC) libraries in ordered microtiter plates required a new approach to screening for clones containing specific DNA sequences. Screening libraries of some 60,000 clones by hybridization to filters prepared from individual 96-well micr ...

Yeast artificial chromosome (YAC) libraries stored in microtiter plates are available for screening as either complex PCR pools or hybridization filters generated from YACs gridded at high densities (see Chapter 3). Different libraries may be available as either PCR pools, hybridiz ...

Telomeres are specialized structures that form the ends of eukaryotic chromosomes and that are required to fulfill a number of varied functions, see Biessman and Mason for recent review (1). First, they must protect the chromosome ends from degradation and from fusion and recombination with ...