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Thông tin chi tiết về Yeast Artificial Chromosomes (YAC)
Yeast Artificial Chromosomes (YAC) was first described by Murray and Szostak in 1983.A YAC can be considered as a functional artificial chromosome, derived from the DNA of the yeast, Saccharomyces cerevisiae, which is then ligated into a bacterial plasmid.YAc vector is used to clone very large DNA fragment to construct genomic library. While a plasmid vector allows the insertion DNA fragment of about 10,000 nucleotide base pairs, YAC vectors allows the insertion of DNA fragments up to 100-500 kb. A YAC vector includes
- TEL: telomere (TEL) is located at each end of chromosome. It protects the linear DNA from degradation by nucleases.
- CEN: The centromere which is the attachment site for mitotic spindle
- ARS: Replication origin sequences which are specific DNA sequences that allows the DNA replication machinery to assemble on the DNA and move at the replication forks.
- A and B: YAC contains two selectable markers that allow the easy isolation of yeast cells that have taken up the artificial chromosome.
o URA3-involved in uracil biosynthesis
o TRP1 -involved in tryptophan biosynthesis
- Recognition site for the two restriction enzymes EcoRI and BamHI.
- Ampr – an ampicillin resistance gene allows positive selection by ampicillin resistance in bacteria
The four main types of yeast plasmids are:
- Yeast Integrating plasmids (YIp): These plasmids lack an ORI and must be integrated directly into the host chromosome via homologous recombination.
- Yeast Replicating plasmids (YRp): These vectors contain an Autonomously Replicating Sequence (ARS) derived from the yeast chromosome. As the name suggests, these vectors can replicate independently of the yeast chromosome; however, they tend to be unstable and may be lost during budding.
- Yeast Centromere plasmids (YCp): These are considered low copy vectors and incorporate part of an ARS along with part of a centromere sequence (CEN). These vectors replicate as though they are small independent chromosomes and are thus typically found as a single copy. Unlike the ARS vectors, CEN vectors are stable without integration.
- Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle (a natural yeast plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors can also be controlled if specific regulatable elements are included
Construction of YAC
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 construction of a YAC library consists of the following steps:
- Ligation of selectable 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.
- Ligation of necessary centromeric sequences for mitotic stability
- 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.
- Ligation of artificial telomeric sequences to convert circular plasmid into a linear piece of DNA
- Insertion of DNA sequence (up to 1000kb)
- Transformation yeast colony
- Yeast expression vectors, such as YACs, YIps (yeast integrating plasmids), and YEps (yeast episomal plasmids) can be used to express eukaryotic proteins that require posttranslational modification.
- YACs vectors are able to insert large fragments of DNA and can be utilized to clone and assemble the entire genomes of an organism.
- the inserted sequences can be cloned and physically mapped using a process called chromosome walking
- Study expression of entire mammalian genes
- YACs are significantly less stable than BACs, producing “chimeric effects”
- Difficult to isolate YACs from host yeast genome
- Have low copy number
- Slow growth rate
- Poor transformation efficiency
By |2018-04-13T10:29:43+00:00April 10th, 2018|Molecular Biology|Comments Off on Yeast Artificial Chromosomes (YAC)
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