#YAC

Restriction Enzyme and Vector

A YAC-derived construct must contain which sequence to survive in yeast?

Functional yeast artificial chromosome must replicate, segregate and be selectable. Autonomously replicating sequence acts as origin where replication complex assembles, licensing DNA duplication once per cell cycle. Without ARS, linear construct fails to duplicate and is lost. Centromere ensures proper segregation, telomeres protect linear ends, but ARS provides essential replication initiation. Bacterial ori functions only in Escherichia coli for shuttling, lac operon regulates bacterial transcription, not yeast survival. Therefore presence of yeast origin ARS is indispensable for autonomous replication and maintenance of YAC construct within Saccharomyces cerevisiae nucleus.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which element distinguishes religated YAC from vector?

YAC cloning involves ligation of insert DNA between two arms each carrying telomere, selection marker and centromere. Linearized vector without insert can recircularize through non-homologous end joining, regenerating empty YAC. SUP4, encoding suppressor tRNA for ochre mutation, serves as distinguishing marker. When SUP4 is interrupted by insert, suppressor phenotype is lost, producing red colonies in ade2-101 background, while religated empty vector retains functional SUP4 giving white colonies. CEN, ARS, URA3, TRP1 present in both forms cannot differentiate. SUP4 inactivation provides positive visual indication of recombinant formation versus vector background.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which statement about YAC is FALSE?

Yeast artificial chromosomes enable cloning of 100 kb to 1 megabase DNA, often maintained singly in yeast and shuttled to Escherichia coli for manipulation. However, characterization reveals significant instability. Because yeast recombination machinery acts on repetitive mammalian DNA, chimerism, internal deletions and rearranged inserts are common, sometimes affecting 20-50 percent of clones. Multiple YACs can coexist per cell and insert can be unstable during mitotic propagation. Therefore claim of complete stability without rearrangements is incorrect. Understanding limitations led to development of BACs and PACs offering greater structural fidelity for genome projects.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which feature makes YAC resemble natural yeast chromosomes?

Yeast artificial chromosomes were designed to replicate and segregate like native yeast chromosomes during mitosis and meiosis. Functionality requires three cis elements: autonomously replicating sequence acting as origin, centromere CEN conferring mitotic stability and ensuring proper spindle attachment and two telomeres protecting linear ends from degradation and fusion. URA3 and TRP1 provide selection, but structural resemblance relies on CEN, TEL and ARS combination. This tripartite core allows YAC to behave as an authentic linear chromosome, carrying megabase inserts and undergoing normal chromosome segregation in Saccharomyces cerevisiae.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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Restriction Enzyme and Vector

Which is a selectable marker in yeast artificial chromosome (YAC)?

Yeast artificial chromosomes require selectable markers functional in auxotrophic yeast strains to ensure maintenance. URA3 encodes orotidine-5'-phosphate decarboxylase involved in uracil biosynthesis, complementing ura3 mutant phenotype. Yeast lacking functional URA3 cannot grow without uracil, so only transformants harboring YAC with URA3 survive on minimal medium. Similar markers include TRP1 for tryptophan and LEU2 for leucine selection. URA3 also allows counter-selection with 5-fluoroorotic acid for manipulating constructs. Bacterial markers like ampR are not selectable in yeast, making eukaryotic auxotrophic markers indispensable.

Ref: NCERT Biology Class XII Principles on Klenow fill-in labeling, Lehninger Chapter 9 DNA cloning techniques, and Molecular Cloning by Sambrook Chapter 10 documenting end-labeling of cohesive termini.

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