75. When circular plasmids having a centromere sequence are transformed into yeast cells, they replicate and segregate in each cell division. However, if a linear chromosome is generated by cutting the plasmid, at a single site with a restriction endonuclease, the plasmids are quickly lost from the yeast. It is known that genes on the plasmids are lost because of the instability of the chromosome ends. What could be done so as to restore its stability and can be inherited?

(1) Methylation of adenine residues of the plasmid.
(2) Complexing the plasmid ends with histone proteins.
(3) By incorporating telomere sequences to the end of plasmid.
(4) By incorporating acetylated histone proteins to the plasmid ends.

Introduction

In yeast genetics, plasmids are widely used as vectors for gene cloning and expression. Circular plasmids containing centromere sequences (CEN) replicate and segregate stably during cell division. However, when these plasmids are linearized by cutting at a single site, they become unstable and are rapidly lost from yeast cells. This instability arises because linear DNA ends resemble chromosome ends that require specialized structures for protection and replication. Understanding how to restore stability to linear plasmids is essential for their effective use in yeast molecular biology.

Why Are Linear Plasmids Unstable in Yeast?

• Chromosome Ends Require Telomeres:
  Natural yeast chromosomes are linear and have telomeres—specialized repetitive DNA sequences at their ends that protect chromosome termini from degradation and fusion.

• Lack of Telomeres on Linearized Plasmids:
  When a circular plasmid is cut to form a linear molecule, it lacks these protective telomeric sequences, making the DNA ends susceptible to degradation, recombination, and loss during mitosis.

• Centromere Alone Is Not Enough:
  Although centromere sequences ensure proper segregation, they cannot compensate for the absence of telomeric protection at chromosome ends.

How to Restore Stability: Incorporation of Telomere Sequences

• Adding Telomeric DNA to Plasmid Ends:
  Incorporating yeast telomere sequences at the ends of linear plasmids mimics natural chromosome ends, providing the necessary protective cap.

• Telomere Function:
  Telomeres recruit telomere-binding proteins that shield DNA ends from exonucleases and prevent recognition as DNA damage. They also facilitate proper replication and segregation.

• Experimental Evidence:
  Studies show that linear plasmids with telomeric repeats (e.g., from yeast or Tetrahymena rDNA termini) are maintained stably during mitosis and meiosis in yeast, whereas linear plasmids without telomeres are rapidly lost.

Why Other Options Are Ineffective

• Methylation of Adenine Residues:
  DNA methylation affects gene expression and DNA-protein interactions but does not provide end protection or replication stability for linear DNA.

• Complexing Ends with Histone Proteins or Acetylated Histones:
  While histones package DNA into chromatin, they do not specifically protect or stabilize free DNA ends lacking telomeric sequences.

Summary Table

Conclusion

The instability of linear plasmids in yeast is primarily due to the absence of protective telomere sequences at their ends. Incorporating yeast telomere sequences at the ends of linearized plasmids restores their stability by mimicking natural chromosome ends, enabling proper replication, protection, and segregation. This approach is essential for maintaining linear plasmids in yeast cells over multiple generations.

Keywords

linear plasmid stability, yeast telomeres, centromere plasmids, telomere sequences, plasmid inheritance, chromosome end protection, telomere-binding proteins, yeast molecular biology, plasmid linearization, DNA end stability

Correct answer:
(3) By incorporating telomere sequences to the end of plasmid.