Yeast Artificial Chromosome (YAC)

What is gene cloning?

Gene cloning is a process of generating large numbers of identical copies of a gene through replication in a host cell, it is the process in which a gene of interest is located, then copied out of DNA extracted from an organism.

Gene cloning

A YAC is an artificially constructed chromosome derived from the DNA of the yeast Saccharomyces cerevisiae. A vector created in the laboratory to clone Deoxyribonucleic acid (DNA) fragments larger than 100 kilo base pair (kbp) and up to 3000 kbp. It is double stranded and linear, it contains the telomeric, centromeric and replication origin sequences needed for the replication and preservation in yeast cells.

DNA

YACs are built using an initial circular plasmid which is broken into two linear molecules using restriction enzymes and then DNA ligase is used to ligate a gene of interest between the two linear molecules to form a single linear piece of DNA, the recombinant DNA.

Using this method, large contiguous segments of DNA from any organism can be cloned into any suitable vector in such a way that the recombinants can be transformed into yeast cells where they are stably propagated.

When discussing YAC, the following must be remembered; Tel, Cen, Ori, A & B, Recognition sites: EcoRI and BamHI

• Tel: Sequences located at each chromosome end which protects the linear DNA from degradation by nucleases.

• Cen: Sequences which constitute the attachment site for mitotic spindle fibers that pull one copy of each duplicated chromosome into each new daughter.

• Ori: These are specific DNA sequences that allow DNA replication machinery to assemble on the DNA and move at the replication forks.

• A & B: these are selectable markers.

• Recognition site for the two restriction enzymes.

Cloning into a YAC

Genomic DNA is partially digested by the restriction enzyme EcoRI and very large DNA fragments are obtained from this digestion.

Drawn by me into my notes

• The YAC is digested by the two restrict enzymes, EcoRI and BamHI

• Genomic DNA fragment and YAC recombine at the EcoRI sites and are covalently bonded by the DNA ligase.

• The resulting product is a recombinant YAC vector, that is, a YAC with genomic DNA inserted.

• This vector can now be used to infect yeast cells to generate unlimited number of copies.

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References

1. https://www.khanacademy.org/science/biology/biotech-dna-technology/dna-cloning-tutorial/a/overview-dna-cloning

2. https://www.ncbi.nlm.nih.gov/m/pubmed/7765076/

3. https://www.medicinenet.com/script/main/art.asp?articlekey=6052

4. https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/yeast-artificial-chromosome

5. https://en.m.wikipedia.org/wiki/Yeast_artificial_chromosome