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 ...

There are many methods describing the preparation of DNA from Saccharomyces cerevisiae. The method described in this chapter is a modification of those described by Olson et al. (1) and Philippsen et al. (2). Although simpler techniques are available (3), this method routinely provides relat ...

Restriction enzyme analysis of yeast artificial chromosome (YAC)-cloned DNA allows direct comparison to genomic DNA, particularly in regions were pulsed-field gel electrophoresis (PFGE) maps are available, or to DNA cloned in other vectors (1,2). Furthermore, it allows the compari ...

The ordering of yeast artificial chromosome (YAC) clones by sequence-tagged site (STS)-content mapping has proven an effective means of constructing large, contiguously cloned arrays of DNA, some of which span almost entire human chromosomes (1). This method requires that each YAC clone ...

Fluorescence in situ hybridization (FISH) is a rapid procedure for mapping YACs on metaphase chromosomes and for identifying chimeric YACs that contain cocloned DNA fragments from different genomic regions.

The quantitative appraisal of the number of foreign gene copies integrated within the genomes of stably transfected cells is most conveniently performed using the strategy known as Southern blotting (1,2). First introduced by E. M. Southern in 1975 (2) the basic protocol involves the follow ...

The short-term expression of DNA introduced into eukaryotic cells is now widely used to investigate the biological activities of cellular and viral genes or their products. A number of different transfection methods are in common use and can be broadly divided into two categories, based on the ...

Bacterially propagated plasmid DNA can be transfected into established eukaryotic cell lines or primary cell cultures by a variety of techniques, such as electroporation (see Chapter 5, this vol) (1), scrape-loading (2), and DEAE dextran (see Chapter 3) or calcium phosphate mediated gene t ...

DNA transfection is one of the most important techniques in molecular genetics. It is this technique that has made possible the dissection of complex eukaryotic genes and the characterization of the function of their components (1–7) as well as the isolation of particular genes on the basis of th ...

When DNA is introduced into eukaryotic cells, it can be integrated into the genome by homologous or illegitimate recombination 1,2. Despite great efforts to gain insight into the molecular mechanisms, our understanding of the recombination process is still in its infancy. In the absence of a m ...

Murine erythroleukemia (MEL) cell lines are erythroid progenitor cells derived from the spleens of susceptible mice infected with the Friend virus complex 1. These virally transformed cells are arrested at the proerythroblast stage of development and can be maintained in tissue cult ...

The study of gene regulation in eukaryotic cells involves a practical requirement for two distinct techniques: first, a transfection system, or more simply, a way of getting DNA into a cell; and second, a reporter system, which, as the name suggests, is a means of finding out what the transfected DNA does f ...

Efforts to expand the current repertory of cell types amenable to transfection have often been thwarted by a common obstacle-namely, the low tolerance displayed by the recipient cells toward the gene-transfer regimen itself. As a result, several laboratories have turned to the use of synth ...