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Faithful segregation of the multicopy yeast plasmid through cohesin-mediated recognition of sisters.

ABSTRACT: The 2-microm yeast plasmid, a benign high-copy nuclear parasite, propagates itself with nearly the same fidelity as the chromosomes of its host. Equal plasmid segregation is absolutely dependent on the cohesin complex assembled at the plasmid partitioning locus STB. However, the mechanism of cohesin action in the context of multiple plasmid copies, resident within two separate clusters after DNA replication, is unknown. By using "single-copy" derivatives of the 2-microm plasmid, we demonstrate that recruitment of cohesin at STB during S phase indeed translates into cohesion between plasmid molecules. Through binary fluorescence tagging, we reveal that segregation of replicated plasmids occurs in a sister-to-sister fashion. Thus, cohesin serves the same fundamental purpose in plasmid and chromosome segregation.

SUBMITTER: Ghosh SK

PROVIDER: S-EPMC1941829 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Faithful segregation of the multicopy yeast plasmid through cohesin-mediated recognition of sisters.

Ghosh Santanu K SK Hajra Sujata S Jayaram Makkuni M

Proceedings of the National Academy of Sciences of the United States of America 20070801 32

The 2-microm yeast plasmid, a benign high-copy nuclear parasite, propagates itself with nearly the same fidelity as the chromosomes of its host. Equal plasmid segregation is absolutely dependent on the cohesin complex assembled at the plasmid partitioning locus STB. However, the mechanism of cohesin action in the context of multiple plasmid copies, resident within two separate clusters after DNA replication, is unknown. By using "single-copy" derivatives of the 2-microm plasmid, we demonstrate t ...[more]

PMID: 17670945

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Project description:BackgroundA commonly used approach to improve recombinant protein production is to increase the levels of expression by providing extra-copies of a heterologous gene. In Komagataella phaffii (Pichia pastoris) this is usually accomplished by transforming cells with an expression vector carrying a drug-resistance marker following a screening for multicopy clones on plates with increasingly higher concentrations of an antibiotic. Alternatively, defective auxotrophic markers can be used for the same purpose. These markers are generally transcriptionally impaired genes lacking most of the promoter region. Among the defective markers commonly used in Saccharomyces cerevisiae is leu2-d, an allele of LEU2 which is involved in leucine metabolism. Cells transformed with this marker can recover prototrophy when they carry multiple copies of leu2-d in order to compensate the poor transcription from this defective allele.ResultsA K. phaffii strain auxotrophic for leucine (M12) was constructed by disrupting endogenous LEU2. The resulting strain was successfully transformed with a vector carrying leu2-d and an EGFP (enhanced green fluorescent protein) reporter gene. Vector copy numbers were determined from selected clones which grew to different colony sizes on transformation plates. A direct correlation was observed between colony size, number of integrated vectors and EGFP production. By using this approach we were able to isolate genetically stable clones bearing as many as 20 integrated copies of the vector and with no significant effects on cell growth.ConclusionsIn this work we have successfully developed a genetic system based on a defective auxotrophic which can be applied to improve heterologous protein production in K. phaffii. The system comprises a K. phaffii leu2 strain and an expression vector carrying the defective leu2-d marker which allowed the isolation of multicopy clones after a single transformation step. Because a linear correlation was observed between copy number and heterologous protein production, this system may provide a simple approach to improve recombinant protein productivity in K. phaffii.

Dataset Information

Faithful segregation of the multicopy yeast plasmid through cohesin-mediated recognition of sisters.

Publications

Faithful segregation of the multicopy yeast plasmid through cohesin-mediated recognition of sisters.

Similar Datasets

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