Yeast Artificial Chromosomes (YACs)

Yeast is a type of single-celled fungus that can grow and reproduce by budding. Yeast is widely used in baking, brewing, and biotechnology. Yeast cells have a nucleus that contains their genetic material, which is organized into linear structures called chromosomes. Chromosomes are made of DNA, the molecule that stores and transmits genetic information.

DNA stands for deoxyribonucleic acid. It is composed of four types of nucleotides: adenine (A), thymine (T), cytosine (C), and guanine (G). These nucleotides form complementary base pairs: A with T and C with G. The nucleotides are arranged in a double helix structure, where two strands of DNA are held together by hydrogen bonds between the base pairs. The sequence of nucleotides in a DNA strand determines the genetic code, which instructs the cell on how to make proteins.

Proteins are the molecules that perform most of the functions in living organisms. They are made of amino acids, which are linked together by peptide bonds. The order of amino acids in a protein is determined by the order of nucleotides in the DNA that encodes it. Each group of three nucleotides called a codon, specifies one amino acid. For example, the codon ATG codes for the amino acid methionine.

The process of making proteins from DNA is called gene expression. It involves two main steps: transcription and translation. Transcription is the synthesis of a messenger RNA (mRNA) molecule from a DNA template. mRNA is a single-stranded molecule that carries genetic information from the nucleus to the cytoplasm, where translation occurs. The translation is the synthesis of a protein from an mRNA template. It involves the use of ribosomes, which are complexes of RNA and protein that read the mRNA and assemble the amino acids into a polypeptide chain.

Yeast cells have about 16 chromosomes that contain about 12,000 genes. Genes are segments of DNA that encode specific proteins or functional RNAs. Some genes are essential for the survival and growth of yeast cells, while others are involved in specialized functions such as metabolism, stress response, or mating. Yeast cells can also exchange genetic material with other yeast cells through sexual reproduction or horizontal gene transfer.

Yeast artificial chromosomes (YACs) are synthetic chromosomes that can be constructed and manipulated in yeast cells. They can carry large segments of foreign DNA from other organisms, such as humans or plants. YACs are useful tools for studying gene function, genome organization, and chromosome behavior in eukaryotes. In this article, we will explore the structure, working principle, process, advantages, uses, and limitations of YACs.

A yeast artificial chromosome (YAC) is a genetically engineered DNA molecule that can replicate and segregate in yeast cells. It is composed of four main elements that are derived from the natural yeast chromosomes:

• Telomeres: These are the sequences at the ends of chromosomes that protect them from degradation and fusion. A YAC has two copies of a yeast telomeric sequence, one at each end of the linear DNA molecule. The telomeres are typically about 300 base pairs in length.

• Centromere: This is a DNA sequence that allows the YAC to be replicated and segregated during cell division. It is typically about 100 base pairs in length. The centromere ensures that each daughter cell receives one copy of the YAC after mitosis or meiosis.

• ARS: This stands for autonomously replicating sequence, which is a DNA sequence where DNA replication begins. A YAC has one copy of a yeast ARS, which acts as an origin of replication for the YAC DNA. The ARS is usually about 1000 base pairs in length.

• Selectable markers: These are genes that confer resistance to certain drugs or nutrients or produce a visible phenotype, such as color or shape. A YAC has two or more selectable markers, one for each arm of the YAC vector. The selectable markers allow the identification and isolation of yeast cells that have taken up the YAC vector or the recombinant YAC with the foreign DNA insert. Some examples of selectable markers are TRP1, URA3, SUP4, and LEU2.

The structure of a YAC vector is shown below:

The foreign DNA fragment to be cloned is inserted into a unique restriction site within one of the selectable markers, such as SUP4. This disrupts the function of the marker gene, resulting in a different phenotype for the recombinant YAC compared to the original YAC vector. For example, if SUP4 is inactivated by foreign DNA insertion, the yeast cells will turn red instead of white.

The size of the foreign DNA fragment that can be inserted into a YAC ranges from 100 to 1000 kilobases (kb), which is much larger than other cloning systems, such as plasmids or cosmids. This makes YACs suitable for cloning and mapping large genomic regions or entire genomes.

The yeast artificial chromosome, which is often shortened to YAC, is an artificially constructed system that can undergo replication. The design of a YAC allows extremely large segments of genetic material to be inserted. Subsequent rounds of replication produce many copies of the inserted sequence in a genetic procedure known as cloning.

The principle of YACs is similar to that of plasmids or cosmids, which are circular DNA molecules that can replicate independently of the host chromosome. The experimenter introduces some typical elements that are necessary for correct replication. In the case of YACs, these elements are the centromeres and telomeres of the yeast chromosomes, which must be inserted into the DNA being cloned.

Centromeres are the regions of the chromosome that attach to the spindle fibers during cell division and ensure proper segregation of the chromosomes. Telomeres are the sequences at the ends of the chromosomes that protect them from degradation and fusion. Both centromeres and telomeres are essential for maintaining the stability and integrity of linear chromosomes.

The constructs can be transformed into yeast cells and are then replicated there. In contrast to the vectors, YACs are not circular; they are made of linear DNA. This allows them to accommodate larger inserts than plasmids or cosmids, which have size limitations due to their circularity.

YACs can carry inserts ranging from 100 kb to 3000 kb (1 kb = 1000 base pairs), which makes them suitable for cloning large genomic regions or even entire genes. For comparison, plasmids can carry inserts up to 15 kb and cosmids up to 45 kb.

YACs can also be used to express eukaryotic proteins that require post-translational modifications, such as glycosylation or phosphorylation. These modifications cannot be performed by bacterial cells, which are often used as hosts for plasmid or cosmid vectors. Yeast cells, being eukaryotes, have the machinery to perform these modifications and thus can produce functional proteins from YACs.

YACs are extremely popular for those trying to analyze entire genomes. They can be used to construct physical maps of chromosomes by assembling contiguous stretches of genomic DNA (YAC contigs). They can also be used to study the functions of genes in the context of flanking sequences by reintroducing YACs intact into mammalian cells where the introduced genes are expressed.

However, YACs also have some limitations and drawbacks. They are prone to chimerism, i.e., containing DNA from different locations in the genome in a single clone. They also 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 cloning with YACs is low (about 1000 clones are obtained per microgram of vector and insert DNA). The yield of YAC DNA isolated from a yeast clone containing a YAC is quite low (only a few percent of the total DNA in the recombinant yeast cell). It is difficult to obtain even 1 μg of YAC DNA. The cloning of YACs is also too complicated to be carried out by a lone researcher.

In summary, YACs are artificial chromosomes that can replicate in yeast cells and carry large inserts of genetic material. They have advantages over other cloning systems in terms of insert size and expression of eukaryotic proteins. However, they also have disadvantages such as chimerism, instability, low efficiency, and low yield.

The process of creating and using yeast artificial chromosomes (YACs) can be summarized as follows:

• A YAC vector is initially propagated as a circular plasmid inside a bacterial host using a bacterial origin of replication sequence.

• The circular plasmid is cut at a specific site using restriction enzymes to generate a linear chromosome with two telomere sites at the terminals.

• The linear chromosome is again digested at a specific site with two arms with different selection markers. The selection markers are usually genes that confer resistance to antibiotics or complement auxotrophic mutations in yeast cells.

• The genomic insert, which is the DNA fragment of interest, is then ligated into the YAC vector using DNA ligase enzyme. The insertion of the genomic insert disrupts a reporter gene, such as SUP4, that can be used to distinguish between recombinant and non-recombinant colonies based on their color.

• The recombinant vectors are transformed into yeast cells and screened for the selection markers to obtain recombinant colonies. The yeast cells also provide the necessary centromere and autonomously replicating sequence (ARS) for the YACs to undergo mitotic replication.

• The YAC clones can be isolated and analyzed for their size, structure, and content using various techniques, such as restriction mapping, PCR, hybridization, and sequencing. The YAC clones can also be used for further applications, such as physical mapping, gene expression, and functional studies.

Yeast artificial chromosomes (YACs) provide the largest insert capacity of any cloning system. They can accommodate DNA fragments of up to one million base pairs in length, which is much larger than other vectors such as plasmids or cosmids. This allows for the cloning and analysis of entire genes or genomic regions that would otherwise be difficult to manipulate.

Yeast expression vectors, such as YACs, YIPs (yeast integrating plasmids), and YEPs (yeast episomal plasmids), have an advantage over bacterial artificial chromosomes (BACs) in that they can be used to express eukaryotic proteins that require post-translational modification. Yeast cells can perform processes such as glycosylation, phosphorylation, and splicing that are essential for the function of many proteins. Bacterial cells lack these capabilities and may produce inactive or toxic proteins.

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. For example, some DNA sequences that contain repeated elements or inverted repeats may be deleted or rearranged in bacteria but not in yeast. Similarly, some genes that are silenced or methylated in bacteria may be expressed normally in yeast.

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. This can help to understand the regulation and interaction of genes in their native environment. For instance, YACs have been used to transfer human genes into mouse cells and create transgenic animals for disease modeling.

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Yeast artificial chromosomes (YACs) have been widely used for various purposes in molecular biology and biotechnology. Some of the main uses of YACs are:

• Studying chromosome behavior: YACs were originally constructed in order to study chromosome behavior in mitosis and meiosis without the complications of manipulating and destabilizing native chromosomes. YACs can be used to investigate the mechanisms of chromosome segregation, recombination, replication, and repair in yeast cells. YACs can also be used to introduce specific mutations or modifications into chromosomes and analyze their effects on chromosome function.

• Mapping and sequencing genomes: YACs representing contiguous stretches of genomic DNA (YAC contigs) have provided a physical map framework for the human, mouse, and even Arabidopsis genomes. YACs can be used to isolate large genomic regions of interest and sequence them using shotgun or long-read methods. YACs can also be used to compare the genomic structure and organization of different species or individuals.

• Cloning and expressing large genes: YACs have the advantage of cloning and expressing very large genes or gene clusters that are difficult or impossible to clone in other vectors. For example, YACs have been used to clone and express the human dystrophin gene, which is over 2 Mb long and causes Duchenne muscular dystrophy when mutated. YACs can also be used to clone and express genes that require complex regulation or splicing in eukaryotic cells.

• Transferring genes into mammalian cells: 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. For example, YACs have been used to transfer human genes into mouse cells and create transgenic mice models for various human diseases. YACs can also be used to transfer genes into human cells for gene therapy or genome editing applications.

Despite their many advantages, YACs have some limitations that make them less suitable for some applications. Some of the major limitations of YACs are:

• Chimerism: 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. This can complicate the process of mapping and sequencing the cloned DNA segments, as well as the analysis of gene function and expression.

• Instability: YAC clones frequently contain deletions, rearrangements, or noncontiguous pieces of the cloned DNA. These can occur during the construction, propagation, or manipulation of YACs, and can result in the loss or alteration of important genetic information. As a result, each YAC clone must be carefully analyzed to be sure that no rearrangements of the DNA have occurred.

• Low efficiency: The efficiency of cloning with YACs is low (about 1000 clones are obtained per microgram of vector and insert DNA). This means that large amounts of DNA are required for generating a representative YAC library and that screening and selecting the desired clones can be time-consuming and labor-intensive.

• Low yield: The yield of YAC DNA isolated from a yeast clone containing a YAC is quite low. The YAC DNA is only a few percent of the total DNA in the recombinant yeast cell. It is difficult to obtain even 1 μg of YAC DNA, which can limit the downstream applications that require large amounts of DNA, such as PCR or Southern blotting.

• Complexity: The cloning of YACs is too complicated to be carried out by a lone researcher. It requires specialized equipment, skills, and protocols, as well as access to yeast strains and vectors. Furthermore, the manipulation and modification of YACs often involve homologous recombination techniques that are not widely available or easy to perform.

These limitations have led to the development of alternative cloning systems, such as bacterial artificial chromosomes (BACs) and fosmid vectors, which offer higher stability, efficiency, yield, and simplicity than YACs. However, YACs still remain useful for cloning extremely large DNA segments that cannot be accommodated by other vectors or for expressing eukaryotic proteins that require post-translational modifications.

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