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Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast, ''Saccharomyces cerevisiae'', which is then ligated into a bacterial plasmid. By inserting large fragments of DNA, from 100–1000 kb, the inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used for the Human Genome Project, however due to stability issues, YACs were abandoned for the use of Bacterial artificial chromosomes (BAC). Beginning with the initial research of the Rankin et al., Strul et al., and Hsaio et al., the inherently fragile chromosome was stabilized by discovering the necessary autonomously replicating sequence (ARS);〔Hsiao, C.-L. & Carbon, J. High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene. … of the National Academy of Sciences(1979〕 a refined YAC utilizing this data was described in 1983 by Murray et al.〔Murray, A. W. & Szostak, J. W. E-Resource Login. Nature (1983).〕 The primary components of a YAC are the ARS, centromere, and telomeres from ''S. cerevisiae''. Additionally, selectable marker genes, such as antibiotic resistance and a visible marker, are utilized to select transformed yeast cells. Without these sequences, the chromosome will not be stable during extracellular replication, and would not be distinguishable from colonies without the vector.〔Ratzkin, B. & Carbon, J. Functional expression of cloned yeast DNA in Escherichia coli. … of the National Academy of Sciences (1977).〕〔Struhl, K., Stinchcomb, D. T., Scherer, S. & Davis, R. W. High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules. Proceedings of the … (1979).〕 ==Construction== A YAC is built using an initial circular DNA plasmid, which is typically cut into a linear DNA molecule using restriction enzymes; DNA ligase is then used to ligate a DNA sequence or gene of interest into the linearized DNA, forming a single large, circular piece of DNA.〔 The basic generation of linear yeast artificial chromosomes can be broken down into 6 main steps: 1. Ligation of selective marker into plasmid vector: this allows for the differential selection of colonies with, or without the marker gene An antibiotic resistance gene allows the YAC vector to be amplified and selected for in ''E. coli'' by rescuing the ability of mutant E. coli to synthesize leucine in the presence of the necessary components within the growth medium. ''TRP1'' and ''URA3'' genes are other YAC vector cloning site for foreign DNA is located within the ''SUP4'' gene. This gene compensates for a mutation in the yeast host cell that causes the accumulation of red pigment. The host cells are normally red, and those transformed with YAC only, will form colorless colonies. Cloning of a foreign DNA fragment into the YAC causes insertional inactivation of the gene, restoring the red color. Therefore the colonies that contain the foreign DNA fragment are red.〔Strachan T. (2011). Human molecular genetics / Tom Strachan and Andrew Read, 4th ed.〕 2. Ligation of necessary centromeric sequences for mitotic stability 3. Ligation of Autonomously Replicating Sequences (ARS) providing an origin of replication to undergo mitotic replication Allows the plasmid to replicate extrachromosomally, but renders the plasmid highly mitotically unstable, and easily lost without the centromeric sequences. 4. Ligation of artificial telomeric sequences to convert circular plasmid into a linear piece of DNA 5. Insertion of DNA sequence to be amplified (up to 1000kb) 6. Transformation yeast colony 〔Burke, D., Carle, G. & Olson, M. Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. science (New York, N.Y.) 236, 806–812 (1987).〕 抄文引用元・出典: フリー百科事典『 ウィキペディア（Wikipedia）』 ■ウィキペディアで「yeast artificial chromosome」の詳細全文を読む スポンサード リンク
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