Yeast Artificial Chromosomes

Abstract

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.

Process for Assembly and Transformation into Saccharomyces cerevisiae of a Synthetic Yeast Artificial Chromosome Containing a Multigene Cassette to Express Enzymes That Enhance Xylose Utilization Designed for an Automated Platform

“…The sequences important for maintaining large cloned DNA as a stable yeast linear chromosome are the yeast autonomously replicating sequence ( ARS1 ) necessary for replication, the centromere ( CEN4 ) DNA sequence for segregation at cell division, and the two telomere-like DNA sequences ( TEL ). 27–29…”

“…27 The centromere and telomere sequences allow maintenance of large cloned DNA as stable, single-copy yeast linear chromosomes. 28,29 The S. cerevisiae yeast strain INVSc1 was transformed with YAC4-SUMO-XI-XKS, and the recombinant strain was evaluated for incorporation of the artificial chromosome and for xylose utilization.…”

Protoplast transformation of Kluyveromyces marxianus

“…Moreover, three markers increased the size of pLHZ626 to 10 kb, which was close to the size of a YAC vector. [28] Calcium is a critical factor in the transformation that it can neutralize the negative charges and stimulate the intake of DNA by protoplasts. [29] To test the influence of calcium on the protoplast transformation in K. marxianus, STC and PEG 8000 solution were supplemented with 10-100 mM CaCl 2 .…”

Transfer of disulfide bond formation modules via yeast artificial chromosomes promotes the expression of heterologous proteins in Kluyveromyces marxianus

“…The expression of heterologous gene loaded by YACs is independent of the host genome, avoiding mutual interference between native genes and loaded modules caused by integration. YACs can be employed for the cloning and manipulation of extremely large DNA inserts (up to 3 Mb) in S. cerevisiae, and larger YACs display increased mitotic stability as they increase in size 22,23 . After exceeding 50 kb in size, YACs exhibit high stability comparable to natural chromosomes, with a loss rate of ~0.3% per generation, which is superior to centromeric plasmids 22,24 .…”