Migration of the yeast linear DNA plasmid from the cytoplasm into the nucleus in Saccharomyces cerevisiae

Abstract

The Kluyveromyces linear plasmids, pGKL1 and pGKL2, carrying terminal protein (TP), are located in the cytoplasm and have a unique gene expression system with the plasmid-specific promoter element termed UCS, which functions only in the cytoplasm. In this study we have developed an in vivo assay system in Saccharomyces cerevisiae which enables the detection of a rare migration of the yeast cytoplasmic plasmid to the nucleus, using a pGKL1-derived cytoplasmic linear plasmid pCLU1. pCLU1 had both the UCS-fused LEU2 gene (a cytoplasmic marker) and the native URA3 gene (a nuclear marker) and therefore its cytoplasmic-nucleo localized could be determined by the phenotypic analysis of the marker. The nuclearly migrated plasmids were often detected as linear plasmids having the telomere sequence of the host yeast at both ends, although circular plasmids were also found. The circular form was produced by the the terminal fusion of pCLU1. Insertion of a Ty element into a nuclearly migrated plasmid was observed, allowing the ROAM-regulated expression of the adjacent nuclearly silent UCS-fused LEU2 gene. The nuclearly located plasmids, whether linear or circular, were less sensitive to UV-mediated curing than pGKL and pCLU1.

Progressive alteration of telomeric sequences at one end of a yeast linear plasmid and its possible association with reduced plasmid stability

“…While the circular form was produced by self-circularization of pCLU1, the linear plasmid pTLU carried host telomeric repeats of about 300±350 bp at both ends (Gunge et al 1995. Although the added telomeric sequences varied in the arrangement of TG 1±3 repeats among the individual pTLU plasmids isolated, those at the two ends of any given pTLUs were identical.…”

“…cerevisiae strains harboring pTLU or pRLU1 were used: K12-PT2 (MATa leu2 ura3 rho + pTLU2), K12-PT3 (MATa leu2 ura3 rho + pTLU2), K12-PT3 (MATa leu2 ura3 rho + pTLU3), XS95-6C-PT101 (MATa rad52 his3 leu2 ura3 trpl can r rho + pTLU101) and K12-U2-S1 (MATa leu2 ura3 rho + pRLU1). K12-PT2, K12-PT3 and K12-U2-S1 are isogenic and were derived from K12-PC (MATa leu2 ura3 rho + pGKL2 pCLU1) (Gunge et al 1995;Takata et al 2000). The circular plasmid pRLU1 (8372 bp) arose spontaneously from pCLU1 (8442 bp) by the loss of a 70-bp stretch from an ITR-terminus through a homologous recombination between short direct repeats within the ITR ends .…”

“…The sporadic migration of pCLU1 into the nucleus can be detected by monitoring repression of the LEU2 gene, which is fused to a promoter that is only expressed in the cytoplasm (UCS-LEU2), and expression of the URA3 gene (which is transcribed by RNA polymerase II in the nucleus), which allows the host cells (leu2 ura3) to grow on the selective medium SC-Ura, but not on SC-Leu. After migration into the nucleus, the plasmid was found to replicate in multiple copies in either a circularized or a telomere-associated linear form, without any requirement for the helper pGKL2 (Gunge et al 1995. The linear form, called pTLU, lacked TP and instead carried 300±350 bp sequences made up of yeast telomeric repeats (TG 1±3 )n, added to the ITR ends.…”

Genetic manipulation of Kluyveromyces lactis linear DNA plasmids: gene targeting and plasmid shuffles

“…However, despite being functionally compartmentalized to the cytoplasm, there is evidence that k1 and k2 can physically enter the nucleus. In vivo attachment of telomeres to k1 hybrids strongly implies migration or transport into and out of the nuclear compartment [20,21]. Probably, k1 and k2 exist in equilibrium between the nucleus and cytoplasm, with most molecules pre-dominantly present in the latter.…”

“…Evidence comes from the failure of conventional nuclear genes, i.e. those activated by upstream activating sequence (UAS) elements and transcribed by RNP II, to be expressed when integrated into plasmid k1 [20,21]. Conversely, k1 and k2 genes cloned with their cognate UCS into conventional yeast nuclear vectors are generally not expressed accurately [19]: transcription by the nuclear RNP initiates and terminates aberrantly [18].…”