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).



Baker M (2011) Synthetic genomes: the next step for the synthetic genome. Nature 473: 403, 405–408.

Barton MC, Hoekstra MF and Emerson BM (1990) Site‐directed, recombination‐mediated mutagenesis of a complex gene locus. Nucleic Acids Research 18: 7349–7355.

Bentley KL, Shashikant CS, Wang W, Ruddle NH and Ruddle FH (2010) A yeast‐based recombinogenic targeting toolset for transgenic analysis of human disease genes. Annals of the New York Academy of Sciences 1207(suppl. 1): E58–E68.

Bradshaw MS, Bollekens JA, Ruddle FH et al. (1995) A new vector for recombination‐based cloning of large DNA fragments from yeast artificial chromosomes. Nucleic Acids Research 23: 4850–4856.

Burke DT, Carle GF, Olson MV et al. (1987) Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806–812.

Cavaliere FM, Scoarughi GL and Cimmino C (2009) Interspecific transfer of mammalian artificial chromosomes between farm animals. Chromosome Research : An International Journal on the Molecular, Supramolecular and Evolutionary Aspects of Chromosome Biology 17: 507–517.

Cross SH, Allshire RC, Mckay SJ, Mcgill NI and Cooke HJ (1989) Cloning of human telomeres by complementation in yeast. Nature 338: 771–774.

Del Portillo HA, Fernandez‐Becerra C, Browman S et al. (2001) A superfamily of variant genes encoded in the subtelomeric region of Plasmodium vivax. Nature 410: 839–842.

Den Dunnen JT, Grootscholten PM, Dauwerse JG et al. (1992) Reconstruction of the 2.4 Mb human DMD‐gene by homologous YAC recombination. Human Molecular Genetics 1: 19–28.

Fisher CR (1969) Enzymology of the pigmented adenine‐requiring mutants of Saccharomyces and Schizosaccharomyces. Biochemical and Biophysical Research Communications 34: 306–310.

Gaida A, Becker MM, Schmid CD et al. (2011) Cloning of the repertoire of individual Plasmodium falciparum var genes using transformation associated recombination (TAR). PloS One 6: e17782.

Gordon JW, Scangos GA, Plotkin DJ, Barbosa JA and Ruddle FH (1980) Genetic transformation of mouse embryos by microinjection of purified DNA. Proceedings of the National Academy of Sciences of the USA 77: 7380–7384.

Grimes B and Cooke H (1998) Engineering mammalian chromosomes. Human Molecular Genetics 7: 1635–1640.

Hsiao CL and Carbon J (1981) Characterization of a yeast replication origin (ars2) and construction of stable minichromosomes containing cloned yeast centromere DNA (CEN3). Gene 15: 157–166.

Jakobovits A, Moore AL, Green LL et al. (1993) Germ‐line transmission and expression of a human‐derived yeast artificial chromosome. Nature 362: 255–258.

Kawakami S, Harashima S, Kobayashi A and Fukui K (2006) Transformation of yeast using bioactive beads with surface‐immobilized yeast artificial chromosomes. Methods in Molecular Biology 349: 61–65.

Kazuki Y, Hoshiya H, Takiguchi M et al. (2011) Refined human artificial chromosome vectors for gene therapy and animal transgenesis. Gene Therapy 18: 384–393.

Kleine M, Jung C, Michalek W, Diefenthal T and Dargatz H (1997) Construction of a MluI‐YAC library from barley (Hordeum vulgare L.) and analysis of YAC insert terminal regions. Genome/National Research Council Canada=Genome/Conseil national de recherches Canada 40: 896–902.

Kouprina N and Larionov V (2008) Selective isolation of genomic loci from complex genomes by transformation‐associated recombination cloning in the yeast Saccharomyces cerevisiae. Nature Protocols 3: 371–377.

Kouprina N, Ebersole T, Koriabine M et al. (2003) Cloning of human centromeres by transformation‐associated recombination in yeast and generation of functional human artificial chromosomes. Nucleic Acids Research 31: 922–934.

Krzywinski M, Wallis J, Gosele C et al. (2004) Integrated and sequence‐ordered BAC‐ and YAC‐based physical maps for the rat genome. Genome Research 14: 766–779.

Larionov V, Kouprina N, Graves J et al. (1996) Specific cloning of human DNA as yeast artificial chromosomes by transformation‐associated recombination. Proceedings of the National Academy of Sciences of the USA 93: 491–496.

Leem SH, Kouprina N, Grimwood J et al. (2004) Closing the gaps on human chromosome 19 revealed genes with a high density of repetitive tandemly arrayed elements. Genome Research 14: 239–246.

Marchuk D and Collins FS (1988) pYAC‐RC, a yeast artificial chromosome vector for cloning DNA cut with infrequently cutting restriction endonucleases. Nucleic Acids Research 16: 7743.

Moreira PN, Pozueta J, Giraldo P, Gutierrez‐Adan A and Montoliu L (2006) Generation of yeast artificial chromosome transgenic mice by intracytoplasmic sperm injection. Methods in Molecular Biology 349: 151–161.

Moreira PN, Pozueta J, Perez‐Crespo M et al. (2007) Improving the generation of genomic‐type transgenic mice by ICSI. Transgenic Research 16: 163–168.

Murray AW and Szostak JW (1983) Construction of artificial chromosomes in yeast. Nature 305: 189–193.

Naesby M, Nielsen SV, Nielsen CA et al. (2009) Yeast artificial chromosomes employed for random assembly of biosynthetic pathways and production of diverse compounds in Saccharomyces cerevisiae. Microbial Cell Factories 8: 45–56.

Neil DL, Villasante A, Fisher RB et al. (1990) Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors. Nucleic Acids Research 18: 1421–1428.

Noskov VN, Chuang RY, Gibson DG et al. (2011) Isolation of circular yeast artificial chromosomes for synthetic biology and functional genomics studies. Nature Protocols 6: 89–96.

Pavan WJ, Hieter P, Reeves RH et al. (1990) Modification and transfer into an embryonal carcinoma cell line of a 360‐kilobase human‐derived yeast artificial chromosome. Molecular and Cellular Biology 10: 4163–4169.

Perez‐Luz S and Diaz‐Nido J (2010) Prospects for the use of artificial chromosomes and minichromosome‐like episomes in gene therapy. Journal of Biomedicine and Biotechnology 2010. doi: 10.1155/2010/642804.

Poggiali P, Scoarughi GL, Lavitrano M, Donini P and Cimmino C (2002) Construction of a swine artificial chromosome: a novel vector for transgenesis in the pig. Biochimie 84: 1143–1150.

Sambrook J and Russell DW (2001) Molecular Cloning : A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

Sanchez CP and Lanzer M (2006a) Analysis and screening of yeast artificial chromosome libraries by filter hybridization. Methods in Molecular Biology 349: 27–32.

Sanchez CP and Lanzer M (2006b) Construction of yeast artificial chromosome libraries from pathogens and nonmodel organisms. Methods in Molecular Biology 349: 13–26.

Sasaki E, Suemizu H, Shimada A et al. (2009) Generation of transgenic non‐human primates with germline transmission. Nature 459: 523–527.

Silverman GA, Green ED, Young RL et al. (1990) Meiotic recombination between yeast artificial chromosomes yields a single clone containing the entire BCL2 protooncogene. Proceedings of the National Academy of Sciences of the USA 87: 9913–9917.

Takahashi R and Ueda M (2010) Generation of transgenic rats using YAC and BAC DNA constructs. Methods in Molecular Biology 597: 93–108.

Tennyson RB, Ebran N, Herrera AE and Lindsley JE (2002) A novel selection system for chromosome translocations in Saccharomyces cerevisiae. Genetics 160: 1363–1373.

Tolmachova T, Simpson K and Huxley C (1999) Analysis of a YAC with human telomeres and oriP from Epstein‐Barr virus in yeast and 293 cells. Nucleic Acids Research 27: 3736–3744.

Further Reading

Becker M, Aitcheson N, Byles E et al. (2004) Isolation of the repertoire of VSG expression site containing telomeres of Trypanosoma brucei 427 using transformation‐associated recombination in yeast. Genome Research 14: 2319–2329.

Cocchia M, Kouprina N, Kim SJ et al. (2000) Recovery and potential utility of YACs as circular YACs/BACs. Nucleic Acid Research 28: e81.

Duncan A and Hadlaczky G (2007) Chromosomal engineering. Current Opinion in Biotechnology 18(5): 420–424.

Gama Sosa M, De Gasperi R and Elder G (2010) Animal transgenesis: an overview. Brain Structure and Function 214(2): 91–109.

Huang D and Koshland D (2003) Chromosome integrity in Saccharomyces cerevisiae: the interplay of DNA replication initiation factors elongation factors and origins. Genes Development 17: 1741–1754.

Krivoruchko A, Siewers V and Nielsen J (2011) Opportunities for yeast metabolic engineering: lessons from synthetic biology. Biotechnology Journal 6(3): 262–276.

Moreira PN, Giraldo P, Cozar P et al. (2004) Efficient generation of transgenic mice with intact Yeast artificial chromosomes by intracytoplasmic sperm injection. Biology of Reproduction 71: 1943–1947.

Nielsen TO, Cossons NH, Zannis‐Hadjopoulos M and Price GB (2000) Circular YAC vectors containing short mammalian origin sequences are maintained under selection as HeLa episomes. Journal of Cell Biochemistry 76: 674–685.

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]