Yeast Artificial Chromosomes (YACs) – Definition, Structure, Construction, Uses

What is Yeast Artificial Chromosomes (YACs)?

Yeast artificial chromosomes (YACs) are the result of a recombinant DNA cloning technique used to isolate and propagate extremely large DNA segments in a yeast host. The YAC cloning system enables the replication of exogenous DNA segments as linear molecules on a significantly larger scale than is possible with bacterial cloning systems. The cloning capacity of YACs is limitless, ranging from less than 100kb to more than 1000kb, which is 30 times that of cos-mids (40 kb) and five times that of bacterial artificial chromosomes (BACs, typically 200-300 kb).

YACs facilitate the process of constructing physical maps of genomes as a series of overlapping cloned segments (also known as con-tig maps) due to their extremely large insert size. In addition, the budding yeast host, Saccharomyces cerevisiae, is a eukaryotic organism that offers a variety of homologous recombination-based techniques for the manipulation of cloned exogenous DNA segments within YACs. Vectors that target homologous recombination to exogenous DNA segments in YACs have been created, allowing for the introduction of specific mutations, reporter sequences, or suitable selectable markers. By means of YAC transgenesis, modified YACs can be transferred back into cells as intact DNA segments for analysis of gene structure and function in cultured cells or experimental animals.

Structure of Yeast Artificial Chromosomes

The structure of YACs consists of four main components:

1. Centromere: A DNA sequence that allows the YAC to be replicated and segregated during cell division. It is typically about 100 base pairs in length.
2. Telomeres: Repeated DNA sequences at the ends of the YAC, which protect the DNA from degradation and ensure the stability of the YAC during cell division. They are typically about 1000 base pairs in length.
3. Selectable marker: A gene that confers resistance to a specific antibiotic or drug, allowing selection of cells that have taken up the YAC.
4. Cloning site: A region of the YAC that allows the insertion of foreign DNA fragments for cloning and manipulation.
5. Autonomously replicating sequence (ARS)/Origin of replication: This is a region of DNA that allows the YAC to replicate independently in yeast cells. It is typically about 1000 base pairs in length.
6. Foreign DNA: The foreign DNA is the DNA that is inserted into the YAC. It can be any length, but it is typically up to 1 million base pairs in length.

Working Principle of Yeast Artificial Chromosomes

YACs operate on the premise that yeast cells are capable of replicating and maintaining their own chromosomes. When a YAC is introduced into a yeast cell, the yeast cell replicates it as though it were its own chromosome. This enables replication and maintenance of the foreign DNA within the yeast cell.

YACs can be used to clone DNA fragments as long as 1 million base pairs. In addition, they are valuable for studying the DNA organisation within chromosomes. Numerous organisms, including humans, rodents, and yeast, have had their genomes cloned using YACs.

The following are the stages involved in the operation of YACs:

1. A chromosome of yeast is isolated and modified. The modification procedure typically involves removing the chromosome’s centromere and telomeres.
2. The foreign DNA is incorporated into the chromosome of the modified yeast. Typically, the foreign DNA is flanked by sequences that are similar to the yeast chromosome. This ensures the precise orientation of foreign DNA integration into the chromosome.
3. In a yeast cell, the modified yeast chromosome is introduced. As if it were its own chromosome, the yeast cell will replicate the YAC.
4. The YAC can then be utilised to examine or manipulate foreign DNA.

YACs are a useful instrument for analysing and manipulating large DNA fragments. Significant advances in our comprehension of genetics and biology have been made with their help.

Construction Process of Yeast Artificial Chromosomes

The process of creating a yeast artificial chromosome (YAC) involves several key steps, including:

1. Isolation of large segments of DNA: The first step in creating a YAC is to isolate a large segment of DNA from the organism of interest. This DNA can be obtained from genomic DNA or cDNA libraries.
2. Preparation of the vector: A YAC vector is a circular piece of DNA that has been engineered to carry the DNA of interest. The vector typically includes a yeast origin of replication, selectable markers, and telomere sequences to stabilize the ends of the chromosome.
3. Digestion of the DNA and the vector: The DNA of interest and the YAC vector are then digested with restriction enzymes. The restriction enzymes cut the DNA and the vector at specific sites, creating sticky ends that are complementary to each other.
4. Ligation of the DNA and the vector: The digested DNA and vector are then ligated together using DNA ligase, which joins the complementary sticky ends of the DNA and the vector. The foreign DNA can be introduced into YAC vectors using a variety of methods, including electroporation and transformation.
5. Transformation of yeast cells: The ligation mixture is then introduced into yeast cells through a process called transformation. The yeast cells take up the vector and incorporate it into their genome. Transformation is a process by which DNA is introduced into a cell. The yeast cells will replicate the YAC as if it were their own chromosome.
6. Selection of transformed cells: After transformation, the yeast cells are plated on a selective medium that allows only cells containing the YAC vector to grow. Yeast cells containing the YAC can be identified with a genetic marker. A genetic marker is a readily identifiable gene. The genetic marker is expressed by yeast cells containing the YAC. Yeast cells containing YACs can be selected using a variety of methods, including antibiotic resistance and nutritional markers.
7. Verification of the YAC: The transformed cells are then screened to verify the presence of the YAC. This can be done by PCR analysis, Southern blotting, or other methods.
8. Maintenance and propagation of the YAC: Once the YAC has been verified, it can be propagated and maintained in the yeast cells like a natural chromosome.

Overall, the process of creating a YAC involves the insertion of large segments of DNA into a circular vector, which is then introduced into yeast cells and integrated into the yeast chromosome. This provides a powerful tool for genetic research and the creation of artificial chromosomes for gene therapy.

Advantages of Yeast Artificial Chromosomes

• Can accommodate large DNA fragments: YACs can carry DNA fragments as large as several hundred kilobases, which makes them useful for studying complex genetic traits or for mapping large segments of the human genome.
• Replicate like natural chromosomes: YACs replicate like natural chromosomes, which means they can be maintained and propagated in a stable manner over many generations. This allows researchers to generate large amounts of DNA for further analysis.
• Allow for manipulation of DNA: YACs can be easily manipulated by adding or deleting specific DNA sequences, making them useful for studying the function of specific genes or regulatory regions.
• Suitable for transgenic studies: YACs can be used to create transgenic animals, allowing researchers to study the effects of specific genetic mutations on the development and function of organisms.
• Can be used for gene therapy: YACs have potential applications in gene therapy, as they can be used to deliver therapeutic genes to cells or tissues that are affected by genetic disorders.
• Large cloning capacity: YACs can clone DNA fragments as long as 1 million base pairs. Thus, they are excellent for cloning complete genes or gene complexes.
• Ease of construction: The fabrication of YACs is relatively straightforward. They are based on yeast chromosomes, which are well-understood and straightforward to manipulate.
• Usefulness for genome cloning: YACs have been used to clone the genomes of numerous organisms, including those of humans, rodents, and yeast. This has allowed for the in-depth study of these organisms’ genomes and the identification of genes implicated in various diseases and processes.
• Eukaryotic cloning: YACs can be used to clone DNA from eukaryotic organisms, which is impossible with plasmids and phage. This makes them excellent for cloning eukaryotic-specific genes.
• Disease gene identification: Identification of disease-related genes YACs have been used to identify genes implicated in diseases. This has led to the creation of new disease diagnostic tests and treatments.
• Large insert capacity: YACs are capable of cloning DNA fragments up to 1 million base pairs in length, which is significantly larger than the size limit for plasmids and phage. Therefore, they are optimal for the cloning of large genes or gene complexes.
• Usefulness for studying chromosome organization: YACs are useful for investigating chromosome organisation because they can be used to examine the DNA organisation within chromosomes. This is essential for comprehending how genes are regulated and chromosomes are passed down.

Limitations of Yeast Artificial Chromosomes

• Instability: YACs are known to be unstable, meaning that they can undergo rearrangements or deletions over time. This instability can be due to factors such as the size of the YAC, the presence of repetitive DNA sequences, or the strain of yeast used to propagate the YAC.
• Complexity: YACs are more complex to work with than other types of vectors, such as plasmids. YACs require specialized yeast strains and techniques for manipulation, which can make them more time-consuming and expensive to use.
• Size limitation: Although YACs can accommodate large fragments of DNA (up to 1 Mb or more), they have a size limit beyond which they become unstable and difficult to work with. This size limit is typically around 300-400 kb, depending on the particular YAC system used.
• Integration: YACs can be difficult to integrate into the genomes of other organisms. This is because YACs often contain yeast-specific DNA sequences that may interfere with the stability or expression of the YAC in the target organism.
• Limited applications: YACs are primarily used for research purposes and have limited applications in biotechnology or medicine. Other types of vectors, such as viral vectors or bacterial artificial chromosomes, may be more suitable for certain applications.
• Low efficiency: YAC cloning is an inefficient technique. A minor proportion of yeast cells transformed with YAC DNA will contain the desired DNA. This is because YACs are large and fragile, making it challenging to insert them into yeast cells.
• Low yield: YAC DNA is challenging to isolate from yeast cells, resulting in a low yield. Typically, the yield of YAC DNA is very low, making it challenging to obtain significant quantities of the desired DNA. This is because YACs are large and fragile, making it challenging to isolate them from yeast cells.
• Chimeric YACs: YACs may contain chimeric DNA, which is DNA that has been spliced from two distinct sources. This can make identifying the desired DNA problematic. Due to the fact that YACs are large and vulnerable, they are susceptible to fragmentation during the cloning process.
• Difficult to sequence: Because YACs are difficult to sequence, identifying the correct DNA sequence can be challenging. The sequencing of YACs is a time-consuming and costly endeavour. Using YACs to sequence a 1 Mb DNA fragment typically takes several weeks and costs several thousand dollars.
• Cost: YAC cloning is a relatively costly procedure. This is because the requisite reagents and equipment are expensive.

Uses of Yeast Artificial Chromosomes

Yeast Artificial Chromosomes (YACs) have a variety of uses in molecular biology research. Some of the most common uses of YACs are:

1. Cloning large DNA fragments: YACs are commonly used to clone and study large DNA fragments, up to 1 million base pairs in length. This allows researchers to study entire genes or gene complexes in their native form.
2. Construction of genomic libraries: YACs are used to construct genomic libraries, which contain a complete set of an organism’s DNA. These libraries can be used to identify and study genes and other genomic elements.
3. Gene mapping: YACs are used in gene mapping studies to identify the location of genes on chromosomes.
4. Gene therapy: YACs can be used as vectors for gene therapy, in which they deliver therapeutic genes to cells to treat genetic disorders.
5. Transgenic animal generation: YACs can be used to create transgenic animals, which are animals that have been genetically modified to contain foreign DNA. This allows researchers to study the function of genes in vivo.
6. Functional genomics: YACs are used in functional genomics studies to study the function of entire sets of genes, or gene networks, in living cells.
7. Studies of chromosome organisation: YACs can be used to investigate the organisation of DNA within chromosomes. This is essential for comprehending how genes are regulated and chromosomes are passed down.
8. Disease gene identification: Identification of disease-related genes YACs have been used to identify genes implicated in diseases. This has led to the creation of new disease diagnostic tests and treatments.
9. Gene expression studies: YACs can be utilised for the investigation of the expression of genes in yeast cells. This can be used to comprehend the regulation and function of genes.
10. Protein production: YACs can be utilised in yeast cells to produce proteins. This can be used to investigate the function of proteins and create new drugs and therapies.
11. Chromosome walking: YACs can be used to “walk” along chromosomes in order to identify genes and other DNA sequences that are located near to one another. This can be utilised to examine the DNA organisation within chromosomes.
12. Cancer research: YACs have been used to study the genetics of cancer and to identify genes that are involved in the development of cancer.
13. Evolutionary biology: YACs have been used to study the evolution of genes and genomes by comparing the structure and sequence of YACs from different species.
14. Agriculture: YACs have been used to study the genetics of crop plants, with the goal of improving crop yield and resistance to pests and diseases.

FAQ

References

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• Peterson, K. R., Zitnik, G., Huxley, C., Lowrey, C. H., Gnirke, A., Leppig, K. A., … Stamatoyannopoulos, G. (1993). Use of yeast artificial chromosomes (YACs) for studying control of gene expression: correct regulation of the genes of a human beta-globin locus YAC following transfer to mouse erythroleukemia cell lines. Proceedings of the National Academy of Sciences, 90(23), 11207–11211. doi:10.1073/pnas.90.23.11207
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