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
Structure of Yeast artificial Chromosome basically resembles a telocentric chromosome. 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 a 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.
First digestion generates a long linear fragment carrying a telomeric sequence at each end. This cut is performed at the HIS3 gene that flaking 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. First selective marker is 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).
Another major application of YACs is in the study of the regulation of gene expression by cis-acting, controlling DNA elements, that are present either upstream or downstream of large eukaryotic genes, after the transfer of these YACs from yeast to mammalian cells.
Recent technological developments allow the transfer of YACs into mouse embryonal stem (ES) cells and the subsequent generation of transgenic mice. Investigators have begun to employ these artificial chromosomes for the in vivo study of multigenic loci in mammalian cells.
Advantages of Yeast Artificial Chromosomes
Main Advantage of the Yeast Artificial Chromosome is, it is similar to the natural chromosomes and all three structural elements (ARS, CEN, and TEL) are present and functional. In addition to that, it provides the largest insert capacity that is enough for large exogenous DNA cloning.
Another important advantage is that faithful duplication of YACs is guaranteed only if other DNA sequences that are incompatible with ARS do not exist on the construct. This point is particularly relevant when unknown DNA inserts are cloned in the YAC vector, as is the case for genomic libraries, in which there could be cryptic or otherwise unknown ARS-like sequences able to interfere with the ARS function.
Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs (yeast episomal plasmids), have advantageous over bacterial artificial chromosomes (BACs). They can be used to express eukaryotic proteins that require post-translational modification.
A major advantage of cloning in yeast, a eukaryote, is that many sequences that are unstable, underrepresented, or absent when cloned into prokaryotic systems, remain stable and intact in YAC clones.
It is possible to reintroduce YACs intact into mammalian cells where the introduced mammalian genes are expressed and used to study the functions of genes in the context of flanking sequences.
Limitations of Yeast Artificial Chromosomes
A problem encountered in constructing and using YAC libraries is that they typically contain clones that are chimeric, i.e., contain DNA in a single clone from different locations in the genome.
YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of the cloned DNA. As a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA have occurred.
The efficiency of YAC cloning is low (about 1000 clones are obtained per microgram of vector and insert DNA).
YACs have been found to be less stable than BACs. However, the genetic and biochemical background of the host cell also plays an important role in determining the stability of YACs. Indeed, several mutations are known to affect YAC stability and segregation together with natural chromosomes.
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