Yeast artificial chromosome (YAC) is a genetically engineered DNA molecule used to clone DNA sequences in yeast cells. It is generally used in connection with the mapping and sequencing of genomes.
Particular Segments of an organism’s DNA, up to one million base pairs in length, can be inserted into YACs. The YACs, with their inserted DNA, are then taken up by yeast cells. As the yeast cells grow and divide, they amplify the YAC DNA, which can be isolated and used for DNA mapping and sequencing.
What is Yeast artificial chromosome (YAC) ?
Yeast artificial chromosome in short YACs is the result of a recombinant DNA cloning methodology to isolate and clone large segments of DNA in a yeast host. As the name suggests it is an artificial chromosome that means is engineered by humans.
YACs were First developed as linear molecules and stabilized by terminal telomeres for cloning, propagating, and manipulating large segments of DNA in yeast cells. The design of the YACs allows large segments of the DNA to be inserted ranging from 100 – 1000 kb.
Yeast artificial chromosome was devised and first reported by David Burke in 1987, then he also reported its potential to use the construct as a cloning vector for large pieces of DNA. Almost immediately, YACs were used in the large-scale determination of genetic sequences, most prominently the Human Genome Project.
The inserted sequences can be cloned and physically mapped using a process called chromosome walking. This is the process that was initially used in the human genome project. However, due to stability issues, YACs were abandoned for the use of bacterial artificial chromosomes (BAC).
Structure Of Yeast Artificial Chromosomes.
The structure of the Yeast artificial Chromosome basically resembles a telocentric chromosome. The left Arm of the YAC vector sequences contains a Functional telomere (TEL), a centromere (CEN), an origin of replication (ARS: autonomously replicating sequence), and a yeast selectable marker.
On the other hand, the Right arm consists primarily of the contiguous segment of cloned exogenous DNA (up to 1000 kb) and a vector sequence containing a second yeast selectable marker and a functional telomere (TER) sequence at the distal end.
Thus, YACs contain all the cis-acting elements (CEN, ARS, and TEL) required for chromosome replication and proper segregation in the yeast host.
Construction of Yeast Artificial Chromosome.
Yeast Artificial Chromosome is constructed using an initial circular DNA plasmid. Once Plasmid DNA was purified that circular DNA plasmid was cut into linear DNA using a restriction enzyme (eg. BamHI). Restriction Enzyme performed two distinct digestions (Cut) on the initial DNA sequence.
“Note: Restriction enzyme digestion is a process in which DNA is Cut at a specific site also called restriction endonuclease activity.”
First digestion generates a long linear fragment carrying a telomeric sequence at each end. This cut was performed at the HIS3 gene that flaked to the two telomeric DNA sequences, which therefore is excised from the plasmid and lost.
Second digestion is performed at the SUP4 gene, within this gene YAC vector cloning site for foreign DNA is located. as a result of the second digestion, two linear fragments are produced as the left and right arm of the future linear YAC.
Due to the formation of left and right arm selective markers are separated. The first selective marker was adjacent to ARS and CEN on the left arm and the second selectable marker is on the right arm.
Large DNA fragments with ends compatible with the cloning site, obtained from the desired genome source by digestion with an appropriate restriction endonuclease, are ligated with phosphatase treated YAC arms, to create a single yeast transforming DNA molecule.
Primary transformants can be selected for complementation of both selectable markers (eg. trp1 and ura3) mutation in the host to ensure the presence of both chromosomal arms.
Uses of Yeast Artificial Chromosome (YACs)
YAC vectors were initially created for the cloning of large exogenous DNA segments in S. cerevisiae but soon became chromosomal-like platforms for a variety of in vivo experiments. YACs generally used to study chromosome behavior in mitosis and meiosis without manipulating and destabilizing native chromosomes.
Applications of YACs range from generating whole DNA libraries of the genomes of higher organisms to identifying essential mammalian chromosomal sequences necessary for the future construction of specialized mammalian artificial chromosomes (MACs).
The availability of YAC libraries has greatly advanced the analysis of genomes previously cloned in cosmid vectors. For example, YAC clones have been used as hybridization probes for the screening of complementary DNA (cDNA) libraries, thus simplifying the characterization of unidentified genes.
One of the most important uses of YACs is the exploitation of their behavior as endogenous chromosomes to explore genomic instability. YACs have been used to develop assays in budding yeast to identify mutants with enhanced rates of gross chromosomal rearrangements (GCRs).
What is meant by yeast artificial chromosomes?
Yeast artificial chromosome (YAC) is a human-engineered DNA molecule used to clone DNA sequences in yeast cells. YACs are often used in connection with the mapping and sequencing of genomes. Segments of an organism’s DNA, up to one million base pairs in length, can be inserted into YACs.
How are yeast artificial chromosomes made?
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.
What are the components of yeast artificial chromosome?
Artificial Chromosomes, Yeast The short arm (∼5 kb) contains four DNA elements derived from YAC vector sequences: a functional telomere (TEL), a centromere (CEN), an origin of replication (ARS), and a yeast selectable marker.
Which selection system is used in a yeast plasmid recombinant?
Explanation: A normal yeast gene that codes for an enzyme involved in amino acid biosynthesis is incorporated into the vector. The host that is selected has a mutated copy of the incorporated plasmid gene.
Which is better BAC or YAC?
BACs can carry inserts of 200–300 kb or less. YAC DNA is difficult to purify intact and requires high concentration for generating a YAC vector system. BAC is easy to purify intact and can be easily constructed. YACs are often chimeric.