Factors affecting the mitotic stability of high-copy-number integration into the ribosomal DNA of Saccharomyces cerevisiae.
Yeast vectors suitable for high-level expression of heterologous proteins should combine a high copy number with high mitotic stability. The pMIRY integrative vector system, based upon targeted integration into the yeast rDNA locus, developed in our laboratory satisfies these criteria. However, insertion of a (foreign) gene drastically reduced its mitotic stability of the resulting vector in comparison to its parent. In this paper we have investigated a number of possible reasons for this reduction in stability. The results demonstrate that plasmid size is an important, but not the only, determinant of mitotic stability. Stable maintenance is only observed when the complete plasmid has a size no larger than that of the rDNA unit (9.1 kb). In addition stability depends upon the nature of the rDNA fragment present in the plasmid, required for targeting its integration. On the other hand, it turned out to be irrelevant for mitotic stability whether the heterologous gene was expressed or not. These findings will be important in the design of a pMIRY vector suitable for industrial production of heterologous proteins.
Lopes
T S
TS
Department of Biochemistry and Molecular Biology, Vrije Universiteit, Amsterdam, The Netherlands.
de Wijs
I J
IJ
Steenhauer
S I
SI
Verbakel
J
J
Planta
R J
RJ
D004271:DNA, Fungal / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D018628:Gene Dosage; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008938:Mitosis; D008970:Molecular Weight; D010957:Plasmids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004271:DNA, Fungal; D004275:DNA, Ribosomal