Yeast Artificial Chromosomes


Yeast artificial chromosomes (YACs) are shuttle‐vectors that can be amplified in bacteria and employed for the cloning and manipulation of large deoxyribonucleic acid (DNA) inserts (up to 3 Mb pairs) in the yeast Saccharomyces cerevisiae. Artificial chromosomes can be conveniently built and modified in yeast cells using in vivo homologous recombination, a novel process known as ‘recombineering’. The capacity of YACs to accommodate large DNA fragments is exploited to clone clusters of genes surrounded by their native DNA context, where regulatory elements are located. This is important for biotechnology, when YACs are used for engineering genetic determinants of new biochemical pathways for production of secondary metabolites and for heterologous protein expression. YACs can be retrofitted with the appropriate selectable markers and transmitted to cells of different organisms allowing the generation of transgenic animals. Finally, YACs are largely employed in the production of full‐scale genomic libraries, for mapping and functional analysis purposes.

Key Concepts:

  • Yeast artificial chromosomes (YACs) represent the top instruments for the study of eukaryotic genomes and for mobilisation of large genetic elements among bacteria and eukaryotes.

  • YACs behave like naturally existing chromosomes, provided that they are of the proper size, showing comparable stability.

  • YACs can be manipulated directly by classical genetic engineering as well as by modern recombineering technology.

  • Properly retrofitted, YACs can be used in many different organisms, for cloning or genome analysis.

  • Chromosomal translocation can be studied using disposable YACs that do not harbour genetic information essential for cell function.

Keywords: ARS; centromere; DNA cloning; genome manipulation; genomic library; recombineering; recombinagenic targeting; telomere; transgenic organism; yeast vector

Figure 1.

Circular map of plasmid vector pYAC3. The picture shows yeast auxotrophic markers (blue), elements responsible for propagation and selection in bacteria (green), chromosomal structural elements (red and violet) and strategic restriction sites.

Figure 2.

Construction of the YAC. (a) A circular YAC vector able to replicate and be selected in E. coli, due to the presence of the bacterial ori origin of replication and the bla gene for ampicillin resistance (marked in green) and be propagated in yeast cells as a linear molecule containing all necessary chromosomal elements: yeast centromere CEN4 (black dot), autonomously replicating sequence ARS1 (violet) and two Tetrahymena telomeric sequences TEL (red arrows) functional in yeast after linearisation with the BamHI restriction endonuclease. The yeast sup4 gene (yellow) contains a cloning site and is used as a colour marker for selection of YACs containing exogenous insert DNA. (b) DNA fragments with ends compatible to the YAC cloning site are prepared from source DNA. After double digestion of the YAC vector, the markers used to select for transformants are separated on two chromosomal arms: trp1 on the left and ura3 on the right arm (blue boxes). (c) Chromosomal arms ligated with exogenous DNA are selected after transformation of an appropriate yeast strain (ura3, trp1 and ade2). Adapted from Burke et al..

Figure 3.

YAC modification by homologous recombination. (a) New selective markers lys2 (green box) and neo (yellow box) are introduced into the ura3 gene present on YAC by one‐step disruption: transformation with disruption cassette (top) or modified right YAC arm (bottom). (b) Inactivation of the ura3 gene allows subsequent modification of the insert DNA by using linearised yeast integrative plasmid containing a functional copy of the ura3 gene and a mutagenised copy of the exogenous DNA fragment. After plasmid integration (pop‐in), two copies of target DNA are present (wild‐type and mutated). A YAC containing only the mutated copy of the exogenous DNA is obtained after homologous recombination and loss of the integrative plasmid carrying the wild‐type equivalent (pop‐out). (c) Reconstruction of two smaller overlapping YACs into a larger recombinant YAC by recombination between homologous regions of the insert DNA (dark green boxes).



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Further Reading

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Sugiyama M, Yamagishi K, Kim YH et al. (2009) Advances in molecular methods to alter chromosomes and genome in the yeast Saccharomyces cerevisiae. Applied Microbioogy and Biotechnology 84(6): 1045–1052.

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Arnak, Remigiusz, Bruschi, Carlo V, and Tosato, Valentina(Jan 2012) Yeast Artificial Chromosomes. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0000379.pub3]