Question

How are YACs constructed?

Answer 1

Hint: YAC stands for yeast artificial chromosomes that are genetically engineered chromosomes. It is derived from the DNA of the yeast. It is formed by using the technique of genetic engineering.

Complete answer-
Yeast artificial chromosomes are human engineering DNA molecules that are used to clone DNA sequences in the yeast cells. They are used in connecting with the mapping and sequencing of genomes. Saccharomyces cerevisiae is used as the bacterial plasmid in this process. Large fragments of DNA of approx. 100 - 1000 kb are inserted sequences that can be cloned and mapped by using the process of chromosome walking.
YAC is built using circular DNA plasmid which is typically cut into linear DNA molecules by using restriction enzymes; DNA ligases which are used to ligate a DNA sequence or gene of interest. The basic structure of YAC is broken down into following steps.
1. Ligation of selectable markers into the plasmid vector- It helps in the differential selection of colonies without the help of marker genes. YAC can be amplified and selected for in E. coli by rescuing the ability of mutant E. coli for synthesis of leucine in presence of growth medium. The vector cloning site is present on the SUP4 gene that is compensated for mutation in yeast host cells. The host cell is red due to accumulation but YAC form only colourless colonies.
2. Ligation of necessary centromeric sequence for mitotic stability.
3. Ligation of Autonomously Replicating Sequence that provides yeast origin of replication and helps in mitotic replication.
4. Ligation of artificial telomeric sequences that helps in conversion of circular plasmid into a linear piece of DNA.
5. Insertion of DNA sequence that has to be amplified upto 1000kb.
6. Transformation of the yeast colony.

NOTE-
The primary components of YAC are ARS, telomere and centromere from S. cerevisiae. Apart from this, selectable marker genes like antibiotic resistance and a visible marker are also needed to select transformed yeast cells. Without these sequences, the chromosome will not be stable in extracellular replication and it cannot be distinguished from the colonies.