Materials and methods for controlling plasmid copy number and st

Details Materials and methods for controlling plasmid copy number and st Materials and methods for controlling plasmid copy number and st

1985-11-18

1990-06-19

Teskin, Robin L.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 691, 4351723, 435257, 935 13, 935 28, 935 37, 935 56, 935 69, C12N 1500, C12P 2100, C12P 2102

Patent

active

049353500

ABSTRACT:
Methods and constructions for controlling the copy number and for maintaining the stability of a plasmid in yeast cells involving inserting into the plasmid a centromere sequence under the transcriptional control of a regulatable promotor. The promoter is not activated when it is desirable to maintain a copy number of one per haploid cell (e.g. during a culture growth phase) but is activated when expression is desired. When the promoter is inactive, the centromere sequence causes the plasmid to behave as a minichromosome, but upon activation of the promoter transcription through the centromere sequence permits an increase in copy number. Multicopy plasmids are selected for by inserting a G418 resistance gene in the plasmid. Medium concentrations of greater than about 400 .mu.g/ml of G418 will select for cells containing multiple copies of the plasmid while lesser concentrations of G418 select for cells containing a single copy of a plasmid containing G418.sup.R gene and against cells containing no copy of the G418.sup.R gene.

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