68. In a genetic assay, randomly generated fragments of yeast DNA were cloned into a bacterial plasmid containing gene ‘X’ essential for yeast viability on minimal media. The recombinant plasmid was used to transform a yeast strain deficient in recombination and lacking ‘X gene. Transformants. which survive on minimal media and form colonies should essentially have:

(1) Yeast centromeric sequence which ensures integrity of the plasmid after transformation.
(2)Enhancers for the essential gene missing in the transformed strain.
(3) A sequence similar to bacterial origin of replication
(4) Yeast autonomous replicating sequence

Context of the Genetic Assay

In the described assay, yeast DNA fragments are cloned into a bacterial plasmid containing an essential yeast gene ‘X’. This recombinant plasmid is used to transform a yeast strain lacking gene ‘X’ and deficient in recombination. Survival on minimal media requires the plasmid to replicate and express gene ‘X’ to complement the deficiency.

Key Requirements for Plasmid Maintenance and Yeast Viability

• Replication Origin:
  For a plasmid to be maintained in yeast, it must replicate autonomously. This requires a yeast autonomous replicating sequence (ARS), which functions as an origin of replication in yeast cells.

• Centromeric Sequence (CEN):
  While centromeric sequences improve plasmid stability by ensuring proper segregation during mitosis, they are not essential for plasmid replication or survival on minimal media.

• Bacterial Origin of Replication:
  This allows plasmid replication in bacteria but not in yeast.

• Enhancers for Essential Genes:
  Enhancers regulate gene expression but do not enable plasmid replication or maintenance.

Why Yeast ARS Is Critical

• ARS sequences recruit the yeast replication machinery, enabling the plasmid to replicate once per cell cycle.

• Without ARS, the plasmid cannot replicate in yeast, leading to loss of the plasmid and inability to complement the essential gene deficiency.

• Plasmids containing ARS but lacking centromeres can replicate but may be unstable; however, they still allow survival under selective conditions.

Supporting Evidence from Literature

• Yeast centromeric plasmids (YCp) contain both ARS and CEN sequences; ARS is necessary for replication, CEN for stability.

• ARS-containing plasmids enable autonomous replication and support yeast viability when complemented with essential genes.

• Centromeric sequences improve mitotic stability but are not strictly required for plasmid replication or survival on minimal media.

Correct Option

(4) Yeast autonomous replicating sequence

Keywords

yeast autonomous replicating sequence, ARS, yeast plasmid replication, plasmid stability, centromeric sequence, yeast gene complementation, minimal media survival, yeast transformation, yeast shuttle vectors

Summary

For yeast transformants to survive on minimal media using a recombinant plasmid carrying an essential gene, the plasmid must replicate autonomously within yeast cells. This replication requires the yeast autonomous replicating sequence (ARS). While centromeric sequences enhance plasmid stability, they are not essential for replication or survival. Bacterial origins function only in bacteria, and enhancers do not substitute for replication origins. Therefore, the presence of ARS is crucial for plasmid maintenance and yeast viability in this assay.

Final answer:
(4) Yeast autonomous replicating sequence