Yeast Artificial Chromosome (YAC)

3 Key excerpts on "Yeast Artificial Chromosome (YAC)"

• 

  eBook - ePub

  Molecular Biology and Biotechnology

  • Ralph Rapley(Author)
  • 2021(Publication Date)
  • Royal Society of Chemistry

    (Publisher)

  E. coli . YACs are linear molecules when propagated in yeast but must be circularised by a short DNA sequence between the tips of the telomeres for propagation in bacteria. When used as a cloning vehicle, the YAC is cleaved with restriction enzymes to generate two telomeric arms carrying different yeast-selectable markers. These arms are then ligated to suitably digested DNA fragments, transformed into a yeast host and maintained as a mini chromosome.

  Figure 6.6

  Schematic diagram of a YAC cloning vector and its use as a cloning system.

  YACs have become indispensable tools for mapping complex genomes such as the human genome

  35

  because they accommodate much larger fragments of DNA than bacteriophage or cosmid cloning systems, thus simplifying the ordering of the human genome library. The complete library can be contained in approximately 10 000 clones, cutting by a factor of five the number of clones required by other vector systems. However, given the huge capacity and stability of these powerful vectors, YACs have been further developed to address much more than genome analysis. YACs are also used as a chassis in which to engineer sections of heterologous genomes in yeast cells by exploiting the extremely accurate homologous recombination system.

  6.3.2.2 Manipulating Mammals

  Once a YAC has been successfully transformed into a yeast cell, the highly efficient homologous recombination system of S. cerevisiae can be exploited in vivo to manipulate extensively both YAC vector sequences (retrofitting) and their inserts.

  36

  For example, homologous recombination can be used to retrofit mammalian-selectable markers into the vector arms and/or introduce specific mutations into any genomic sequence carried in a YAC, thus generating artificial chromosomes that can be used in the production of transgenic mice (see Figure 6.7 ). A linear DNA fragment consisting of neomycin resistance and LYS2 genes sandwiched between the 5′ and 3′ ends of the URA 3 gene can be targeted into the URA

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Genetic Analysis

  Principles, Scope and Objectives

  • John R. S. Fincham(Author)
  • 2009(Publication Date)
  • Wiley-Blackwell

    (Publisher)

  The ligated mixture is introduced (see Table 3.2) into yeasr cells which are then challenged to grow under conditions that require both the YAC marker genes. The colonies that grow harbour YACs, most of which, if the ligation has been performed with an appropriate ratio of arms to fragments, will carry inscrts. Fragments of DNA of the order of hundreds of kilobases can be doned in this way. In fact, YACs with very long inserts behave more like ordinary chromosomes, as judged by their regularity of partition between daughter cells, than those which carry lesser amounts of DNA to pad out the minimal YAC arms. The success of the YAC strategy is witness to a very remarkable and apparently rather general property of eukaryotic cells - that of being able nor only to assimilate large pieces of DNA across 82 Chapter 3 Screening DNA libraries for functional genes Cenomic libraries and cDNA libraries A collection of DNA fragments cloned from some particular organism is called a library or, some- times, a bank. If the library is intended to include the whole genome it will usually be constructed with a cosmid vector or, even bener, a YAC system, in order to minimize the large number of clones required. Ir is possible to make an approxi- mate calculation of the required number. Suppose, for example, one wanted a complete Saccharomyces library, consisting of cosmid clones with inserts averaging 45 kb. Since the yeast genome contains about 18000 kb it could, ideally, be packed into about 400 cosmids. But that does not allow for the fact that, in a random assemblage of fragments, some sequences will be represented more than once and others not at all. To reduce the probability of any particular bit of the genome being missed to less than 1%, one would need about 2000 items in a random cosmid library of yeast DNA. The chance of recovering a complete functional gene, rather than just a fragment of it, depends on the method used to generate the large fragments.

  Sign up to read

  Learn more about book
• 

  eBook - PDF

  Gene Manipulations in Fungi

  • J.W. Bennett(Author)
  • 2012(Publication Date)
  • Academic Press

    (Publisher)

  Rather, it is the combination and synergism of classical and recombinant genetics that has given Saccharomyces its preeminent position as a powerful and flexible experimental organism. This crucial interplay between classical and modern approaches will be a recurring theme throughout this chapter. II. NEOCLASSICAL GENETICS An important foundation for either classical or recombinant genetics is a well-defined and -described genetic map. The map of the yeast genome satisfies this requirement since there are well over 300 mapped loci including centromere-linked markers for all but one chromosome (Mortimer and Schild, 1980). The nuclear cytology of yeast is too crude to be useful in making a cytogenetic map of yeast chromosomes. Therefore the map has been derived by measuring map distances between genetic markers as a function of recombination frequencies. The yeast haploid genome consists of 14,000 kilobase pairs (kb) of DNA (Lauer et al., 1977) and is genetically defined as containing approximately 4600 cen-timorgans (cM). The correspondence between physical distance and map dis-tance ranges from 2.7 kb/cM on average for chromosome III (Strathern et al., 1979) to approximately 10 kb/cM for a rather limited region of the genome (Shalit et al., 1981). Thus, yeast is endowed with a rather large amount of recombination per base pair in comparison to Drosophila melanogaster, which has tenfold more DNA and a total of 284 cM in the entire genome (Lindsley and Grell, 1968). This relatively high level of recombination may contribute to the ease with which homologous DNA interactions are observed in yeast in com-parison to organisms with larger genomes. A. Isolation of Mutations Construction of a sophisticated map requires large numbers of easily scorable genetic markers. For organisms capable of growth on a defined medium, aux-otrophic mutations are easily isolated and characterized both genetically and biochemically.

  Sign up to read

  Learn more about book