Site-specific recombination of the circular 2 microns-like plasmid pKD1 requires integrity of the recombinase gene A and of the partitioning genes B and C

Abstract

In the circular plasmid pKD1, which stably replicates in Kluyveromyces lactis, the three open reading frames encode a site-specific recombinase (gene A) and two proteins involved in mitotic stability (genes B and C). A recombination analysis of plasmids in which gene B or C is inactivated reveals that unlike the 2,m plasmid of Saccharomyces cereisiae, these genes are also required for the site specificity of plasmid recombination.pKDl is a circular plasmid isolated from Kluyveromyces drosophilarum (7) which is able to replicate stably in Kluyveromyces lactis (3) and other related Kluyveromyces species (6). pKDl belongs to a family of yeast plasmids that includes the 2,um circle of Saccharomyces cerevisiae and other plasmids from the osmophilic yeasts of the Zygosaccharomyces genus. The stable maintenance of yeast plasmids in the cell population is possible because of efficient mitotic partitioning and copy number amplification. These yeast plasmids do not exhibit sequence homologies, but they have very similar genomic organizations and the functions of plasmid genes are analogous (16). In fact, in all plasmids analyzed, one gene codes for a site-specific recombinase while two other genes are involved in plasmid partitioning. Other plasmid elements, i.e., the replication origin, the inverted repeats, and a cis-acting stability locus (4, 9, 10), are required for plasmid replication, recombination, and partitioning, respectively. The site-specific recombinase (FLP) of the 2ji.m plasmid has been studied both in vivo and in vitro (1, 5) and has been shown to be involved in copy number amplification (8,15).In this work, I report an analysis of the stability, copy number, and recombination in K lactis strains of an isomorphic set of vectors containing pKD1 genes which have been inactivated at the level of translation by frameshift mutations. The results revealed a new function of pKD1 partitioning genes in site specificity of recombination, which is the first important difference between pKD1 and the other circular plasmids.The vectors used in this study were derived from pS13 ( Fig.

A DNA replication origin and a replication fork barrier used in vivo in the circular plasmid pKD1

“…The role that Fob1p plays in each of these activities has not been de®ned, but it has been suggested that it acts to facilitate the binding of dierent proteins to each of the cis-acting sites. In pKD1, the B and C gene products, which interact with the CSL to promote ecient plasmid segregation, are also required to act with the product of the A gene, a site-speci®c recombinase, to mediate plasmid ampli®cation (Bianchi 1992). The site-speci®c recombinase acts at sites within the inverted repeats of pKD1, IR1 and IR2.…”

“…The B and C products, together with a 242-bp pKD1 sequence called CSL (cis-acting stability locus), have been shown to mediate stable segregation of the pKD1 circular plasmid in K. lactis (Bianchi et al 1991). Unlike the 2lm plasmid, where the FLP protein alone is sucient for site-speci®c recombination (Som et al 1988), the genes B and C, which are required for plasmid partitioning, are also directly involved in pKD1 recombination (Bianchi et al 1991;Bianchi 1992).…”

Genetics and Molecular Physiology of the Yeast Kluyveromyces lactis

“…In analogy to the 2-m plasmid of S. cerevisiae, pKD1 is phenotypically cryptic and exists as two isomers that can be interconverted by the gene A product, an FLP-like site-specific recombinase. Genes B and C probably code for pKD1 partitioning factors, and autonomous replication requires a cis-acting ori sequence (Bianchi, 1992;Bianchi et al, 1991). pKD1 can be used to transform S. cerevisiae cir 0 strains but plasmid maintenance is very low; vice versa, 2-m-based vectors get lost readily in K. lactis without maintaining constant selective pressure.…”

Inducible Amplification of Gene Copy Number and Heterologous Protein Production in the Yeast Kluyveromyces lactis

“…pKD1 carries a replication origin, the two B and C genes involved in partitioning, a cis-acting partitioning locus (CSL) (5), a gene encoding a site-specific plasmid recombinase (A), and recombinase target sites in the IRs. Site-specific recombination in pKD1 requires the B and C gene products (6). Homologous recombination occurs frequently in K. lactis among plasmid circles (4).…”