The yeast 2-micron plasmid Rep2 protein has Rep1-independent partitioning function.

Equal partitioning of the multi-copy 2-micron plasmid of the budding yeast Saccharomyces cerevisiae requires association of the plasmid Rep1 and Rep2 proteins with the plasmid STB partitioning locus. Determining how the Rep proteins contribute has been complicated by interactions between the components. Here, each Rep protein was expressed fused to the DNA-binding domain of the bacterial repressor protein LexA in yeast harboring a replication-competent plasmid that had LexA-binding sites but lacked STB. Plasmid transmission to daughter cells was increased only by Rep2 fusion expression. Neither Rep1 nor a functional RSC2 complex (a chromatin remodeler required for 2-micron plasmid partitioning) were needed for the improvement. Deletion analysis showed the carboxy-terminal 65 residues of Rep2 were required and sufficient for this Rep1-independent inheritance. Mutation of a conserved basic motif in this domain impaired Rep1-independent and Rep protein/STB-dependent plasmid partitioning. Our findings suggest Rep2, which requires Rep1 and the RSC2 complex for functional association with STB, directly participates in 2-micron plasmid partitioning by linking the plasmid to a host component that is efficiently partitioned during cell division. Further investigation is needed to reveal the host factor targeted by Rep2 that contributes to the survival of these plasmids in their budding yeast hosts.

Use of Yeast Plasmids: Transformation and Inheritance Assays.

The use of the budding yeast Saccharomyces cerevisiae as a model genetic organism has been facilitated by the availability of a wide range of yeast shuttle vectors, plasmids that can be propagated in Escherichia coli and also in yeast, where they are stably maintained at low- or high-copy number, depending on the plasmid system. Here we provide an introduction to the low-copy (ARS/CEN) and multi-copy (2-µm-based) plasmids, the marker genes commonly used for plasmid selection in yeast, methods for transforming yeast and monitoring plasmid inheritance, and tips for working with yeast transformants.

The yeast 2-µm plasmid Raf protein contributes to plasmid inheritance by stabilizing the Rep1 and Rep2 partitioning proteins.

The yeast 2-µm plasmid is a remarkable genetic parasite, managing efficient maintenance at high-copy number with minimal impact on the host. Equal partitioning of the plasmid upon host cell division requires plasmid proteins Rep1 and Rep2 and the plasmid STB locus. The Rep proteins and the plasmid-encoded Raf protein also regulate plasmid gene transcription. In this study, protein interaction assays, sequence analyses and mutational approaches were used to identify domains and residues in Rep2 and Raf required for association with Rep1 and Rep2 and to delineate the Rep2 DNA-binding domain. Rep2 and Raf displayed similarities in interactions with Rep1 and Rep2, in having Rep1 promote their STB association in vivo, and in stabilizing Rep protein levels. Rep2 mutants impaired for self-association were competent for transcriptional repression while those deficient for Rep1 association were not. Surprisingly, Rep2 mutants impaired for either Rep1 interaction or self-association were able to maintain efficient plasmid inheritance provided Raf was present and competent for Rep protein interaction. Our findings provide insight into the Rep protein complexes required for partitioning and transcriptional repression, and suggest that in addition to its transcriptional function, Raf stabilization of Rep partitioning proteins contributes to the remarkable persistence of the 2-µm plasmid.

The 2 microm plasmid causes cell death in Saccharomyces cerevisiae with a mutation in Ulp1 protease.

The 2 microm circle plasmid confers no phenotype in wild-type Saccharomyces cerevisiae but in a nib1 mutant, an elevated plasmid copy number is associated with cell death. Complementation was used to identify nib1 as a mutant allele of the ULP1 gene that encodes a protease required for removal of a ubiquitin-like protein, Smt3/SUMO, from protein substrates. The nib1 mutation replaces conserved tryptophan 490 with leucine in the protease domain of Ulp1. Complete deletion of ULP1 is lethal, even in a strain that lacks the 2 microm circle. Partial deletion of ULP1, like the nib1 mutation, results in clonal variations in plasmid copy number. In addition, a subset of these mutant cells produces lineages in which all cells have reduced proliferative capacity, and this phenotype is dependent upon the presence of the 2 microm circle. Segregation of the 2 microm circle requires two plasmid-encoded proteins, Rep1 and Rep2, which were found to colocalize with Ulp1 protein in the nucleus and interact with Smt3 in a two-hybrid assay. These associations and the observation of missegregation of a fluorescently tagged 2 microm circle reporter plasmid in a subset of ulp1 mutant cells suggest that Smt3 modification plays a role in both plasmid copy number control and segregation.

Molecular and cytogenetic analysis of the telomeric (TTAGGG)n repetitive sequences in the Nile tilapia, Oreochromis niloticus (Teleostei: Cichlidae).

The majority of chromosomes in Oreochromis niloticus, as with most fish karyotyped to date, cannot be individually identified owing to their small size. As a first step in establishing a physical map for this important aquaculture species of tilapia we have analyzed the location of the vertebrate telomeric repeat sequence, (TTAGGG)n, in O. niloticus. Southern blot hybridization analysis and a Bal31 sensitivity assay confirm that the vertebrate telomeric repeat is indeed present at O. niloticus chromosomal ends with repeat tracts extending for 4-10 kb on chromosomal ends in erythrocytes. Fluorescent in situ hybridization revealed that (TTAGGG)n is found not only at telomeres, but also at two interstitial loci on chromosome 1. These data support the hypothesis that chromosome 1, which is significantly larger than all the other chromosomes in the karyotype, was produced by the fusion of three chromosomes and explain the overall reduction of chromosomal number from the ancestral teleost karyotype of 2n=48 to 2n=44 observed in tilapia.