Extrachromosomal replication of shuttle vectors in Dictyostelium discoideum.

We cloned a 12.3-kilobase (kb) endogenous plasmid, Ddp1, found in several wild-type and laboratory strains of Dictyostelium discoideum into pBR322. The cloned plasmids have been used to cotransform D. discoideum cells with B10S, a transformation vector carrying a gene fusion conferring resistance to G418. Whereas B10S DNA alone appears to integrate in a tandem array, the cloned Ddp1 plasmids replicate extrachromosomally and are stably maintained in the absence of selection with an average copy number of 50 to 100 copies per cell. The Ddp1-derived plasmids can be directly recovered by transforming Escherichia coli with bulk nuclear DNA from these cells. Preliminary deletion analysis indicates that not all regions of Ddp1 are necessary for stable replication in D. discoideum. Several recombinant vectors which replicate extrachromosomally in D. discoideum were also isolated. One contains the Act6-neor gene fusion from B10S recombined into one of the cloned derivatives of Ddp1 and can be used to directly transform D. discoideum amoebae, selecting for G418 resistance. Another recombinant is only 5.6 kb and resulted from a deletion of a 16.6-kb cloned Ddp1 hybrid plasmid. An analysis of the vector DNAs present in clones derived from single D. discoideum transformants is also described.     

Nuclear plasmids in the Dictyostelium slime molds

Cellular slime molds are one of only three types of eukaryotes known to contain circular nuclear plasmids. Unlike the 2-microns circle in Saccharomyces, different strains of Dictyostelium can carry different, nonhomologous plasmids. Covalently closed, circular DNA plasmids have been identified in D. discoideum, D. mucoroides, D. giganteum, and D. purpureum. These plasmids range in size from 1.3-27 kb and in copy number from 50-300 molecules per cell. Plasmids have been identified in approximately one-fifth of all isolates examined. The organization of their DNA in nucleosomes establishes their presence in the nucleus. We have successfully cotransformed endogenous Dictyostelium plasmids into D. discoideum using the G418 resistance shuttle vector B10S. Transformants carrying D. discoideum plasmids are recovered at much higher frequency than those carrying plasmids from the other Dictyostelium species. We have constructed recombinant plasmids based on the D. discoideum plasmid Ddp2 and the G418 resistance gene. With these extrachromosomal vectors, transformed cells are recovered at frequencies of up to 10(-4) per input cell, the vectors are stably maintained at high copy number in the absence of selection, and the vectors can be used to introduce foreign DNA sequences into D. discoideum cells.     

Establishment of the nuclear location of Dictyostelium discoideum plasmids

The nuclear location of the Dictyostelium discoideum plasmids was studied using a biochemical approach based on the presence of plasmid sequences in nucleosomes. This analysis revealed that all four of the known plasmids (Ddp1, Ddp2, Ddp3, Ddp5) are present in chromatin. This evidence establishes that the D. discoideum plasmids are not cytoplasmic but located in the nucleus. D. discoideum is unique among eukaryotes in possessing a group of nonhomologous endogenous plasmids in its nucleus. These plasmids are excellent starting material for construction of nuclear transformation and expression vectors. Such vectors upon transformation into D. discoideum are also present in chromatin as expected for DNA located in the nucleus.     

Construction of an extrachromosomally replicating transformation vector for Dictyostelium discoideum

An extrachromosomally replicating transformation vector for Dictyostelium discoideum has been constructed using sequences of the endogenous Dictyostelium plasmid Ddp2. This transformation vector pnDeI (9.6 kb) replicates as a high copy number plasmid in Dictyostelium and is located in the nucleus. It has been constructed as shuttle vector containing the Escherichia coli vector pUC19 for replication and selection in E. coli and a part of the Tn903 transposon which confers resistance to G418 for selection in Dictyostelium. In order to show that the vector can be used for cloning and stable propagation of Dictyostelium DNA, a fragment of the Dictyostelium alpha-actinin gene that was marked with a synthetic oligonucleotide was cloned into pnDeI and found to be stably maintained in the extrachromosomal vector without undergoing noticeable recombination with the endogenous gene.     

Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418.

A protocol that allows the rapid isolation and growth of large numbers of independent G418-resistant Dictyostelium discoideum transformant colonies on the surface of agar media with live bacteria was developed. Transformants grown under these conditions form normal fruiting bodies. Discovery that aggregation of nontransformants was inhibited at a nonselective level of G418 (25 to 35 micrograms/ml) led to the development of a vector maintenance assay. Using this assay we examined the stability of recombinant plasmids derived from the D. discoideum native plasmids Ddp1 and Ddp2. We conclude that the origin of replication of plasmid Ddp1 does not alone confer stable maintenance and thus, Ddp1 must bear additional sequences required for its own maintenance. Analysis of the maintenance of vectors derived from Ddp2 showed that autonomously replicating shuttle vectors that contained bacterial plasmid DNA and from which one element of the Ddp2 inverted repeat was removed were much less stable than vectors that contained a complete inverted repeat or that did not carry a bacterial plasmid. Sequences between the 3' end of the rep gene and the inverted repeat appear to play a role in plasmid maintenance. An intact rep gene and one copy of the inverted repeat element were required for extrachromosomal replication. Maintenance of extrachromosomal vectors was found to be strain dependent. Four traits distinguishing integrating vectors from those capable of autonomous replication were identified.     

Plasmid maintenance functions encoded on Dictyostelium discoideum nuclear plasmid Ddp1.

All of the plasmid-carried genes expressed during vegetative growth are essential for long-term maintenance of plasmid Ddp1 in the nucleus of Dictyostelium discoideum. Deletion of Ddp1 genes expressed only during development had no detectable effect on plasmid maintenance. Deletion of vegetatively expressed genes, either singly or in pairs, resulted in (i) a rapid loss of plasmid from cells grown in the absence of selection for plasmid retention, (ii) variation in the proportion of monomer to multimer forms of the plasmid molecules, and/or (iii) abnormalities in plasmid copy number. At least two plasmid-encoded gene products influence patterns of expression of plasmid genes.     

The ble gene of Streptoalloteichus hindustanus as a new selectable marker for Dictyostelium discoideum confers resistance to phleomycin

An expression cassette has been constructed which allows expression of the ble gene isolated from Streptoalloteichus hindustanus in Dictyostelium discoideum. This construct has been shown to confer resistance to the bleomycin related antibiotic phleomycin. since the uptake of phleomycin by the cells is pH dependent, we established conditions that allow selection of phleomycin-resistant transformants. Vectors pfeI and pfeII contain, in addition to the cassette, a 592 bp fragment of the D. discoideum plasmid Ddp2 that enables the plasmids to replicate extrachromosomally in Dictyostelium when transformed into a strain that expresses a Ddp2-specific transacting factor (12). pfeI and pfeII contain various unique restriction enzyme sites for cloning. They differ in the G/C-content of the sequence upstream of the ATG start codon.     

Propagation of an amplifiable recombinant plasmid in Saccharomyces cerevisiae: flow cytometry studies and segregated modeling

Efficient expression of a foreign protein product by the yeastSaccharomyces cerevisiaerequires a stable recombinant vector present at a high number of copies per cell. A conditional centromere yeast plasmid was constructed which can be amplified to high copy number by a process of unequal partitioning at cell division, followed by selection for increased copy number. However, in the absence of selection pressure for plasmid amplification, copy number rapidly drops from 25 plasmids/cell to 6 plasmids/cell in less than 10 generations of growth. Copy number subsequently decreases from 6 plasmids/cell to 2 plasmids/cell over a span of 50 generations. A combination of flow cytometric measurement of copy number distributions and segregated mathematical modeling were applied to test the predictions of a conceptual model of conditional centromereplasmid propagation. Measured distributions of plasmid content displayed a significant subpopulation of cells with a copy number of 4-6, evenin a population whose mean copy number was 13.5. This type of copy number distribution was reproduced by a mathematical model which assumes that amaximum of 4-6 centromere plasmids per cell can be stably partitionedat cell division. The model also reproduces the observed biphasic kinetics of plasmid number instability. The agreement between simulation and experimental results provides support for the proposed model and demonstrates the utility of the flow cytometry/segregated modeling approach for the study of multicopy recombinant vector propagation.     

The sequence and organization of Ddp2, a high-copy-number nuclear plasmid of Dictyostelium discoideum

The complete nucleotide sequence of the plasmid Ddp2 found in the nucleus of the simple eukaryote Dictyostelium discoideum is reported. This 5852-bp plasmid contains a 2661-bp open reading frame (ORF), named the "Rep gene," and 501-bp imperfect inverted repeats. A 1762-bp section of Ddp2, which includes one of the 501-bp repeat sequences, could be deleted without abolishing extrachromosomal replication. Deletion of the second 501-bp repeat, or interruption of the Rep gene, removed the ability to replicate extrachromosomally. We suggest that Ddp2 encodes a protein, "REP," that positively regulates replication initiation, a regulatory mechanism different from that of the yeast 2 mu plasmid which also possesses inverted repeat sequences. Ddp2 has a structure similar to that of plasmid pDG1, found in an unidentified isolate of Dictyostelium, with a similar sized ORF and inverted repeats. A common evolutionary origin is suggested by considerable sequence homology between the ORFs of pDG1 and Ddp2.     

Maintenance of some ColE1-type plasmids in chemostat culture

Summary When cells carrying the plasmids RP1, pDS4101 (a ColK derivative) or pDS1109 (a ColE1 derivative) were maintained in chemostat culture in the absence of antibiotic selection, plasmid-free segregants were not detected after 120 generations of nutrient-limited growth. By contrast, plasmid-free segregants of pMB9- and pBR322-containing cells arose after approximately 30 generations, irrespective of the host genetic background. However, even though pDS1109 was maintained its copy-number fell five-fold during 80 generations of limited growth. It is suggested that loss of pBR322 occurs following a similar copy-number decrease which results in defective segregation of the plasmid to daughter host cells. This defective segregation was not complemented in trans by either RP1 or pDS4101.     

Adaptive plasmid evolution results in host-range expansion of a broad-host-range plasmid

Little is known about the range of hosts in which broad-host-range (BHR) plasmids can persist in the absence of selection for plasmid-encoded traits, and whether this "long-term host range" can evolve over time. Previously, the BHR multidrug resistance plasmid pB10 was shown to be highly unstable in Stenotrophomonas maltophilia P21 and Pseudomonas putida H2. To investigate whether this plasmid can adapt to such unfavorable hosts, we performed evolution experiments wherein pB10 was maintained in strain P21, strain H2, and alternatingly in P21 and H2. Plasmids that evolved in P21 and in both hosts showed increased stability and decreased cost in ancestral host P21. However, the latter group showed higher variability in stability patterns, suggesting that regular switching between distinct hosts hampered adaptive plasmid evolution. The plasmids evolved in P21 were also equally or more stable in other hosts compared to pB10, which suggested true host-range expansion. The complete genome sequences of four evolved plasmids with improved stability showed only one or two genetic changes. The stability of plasmids evolved in H2 improved only in their coevolved hosts, not in the ancestral host. Thus a BHR plasmid can adapt to an unfavorable host and thereby expand its long-term host range.     

Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryote Dictyostelium discoideum. We have identified two functional domains, a large open reading frame (Rep gene) and a 626-bp fragment containing an origin of replication (ori). The ori, when cloned into a shuttle vector, confers stable extrachromosomal replication in D. discoideum, provided that the Rep gene, which acts in trans, is integrated into the host genome. Ddp2 carries a 501-bp imperfect inverted repeat, and part of the ori overlaps with one of these repeats. The ori sequence contains two direct repeats of 49 bp comprising two 10-bp "TGTCATGACA" palindromes separated by a poly(T.A) sequence. Deletion of either 49-bp repeat abolished extrachromosomal replication.     

Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors

A 15-kb cryptic plasmid was obtained from a natural isolate of Rhodopseudomonas palustris. The plasmid, designated pMG101, was able to replicate in R. palustris and in closely related strains of Bradyrhizobium japonicum and phototrophic Bradyrhizobium species. However, it was unable to replicate in the purple nonsulfur bacterium Rhodobacter sphaeroides and in Rhizobium species. The replication region of pMG101 was localized to a 3.0-kb SalI-XhoI fragment, and this fragment was stably maintained in R. palustris for over 100 generations in the absence of selection. The complete nucleotide sequence of this fragment revealed two open reading frames (ORFs), ORF1 and ORF2. The deduced amino acid sequence of ORF1 is similar to sequences of Par proteins, which mediate plasmid stability from certain plasmids, while ORF2 was identified as a putative rep gene, coding for an initiator of plasmid replication, based on homology with the Rep proteins of several other plasmids. The function of these sequences was studied by deletion mapping and gene disruptions of ORF1 and ORF2. pMG101-based Escherichia coli-R. palustris shuttle cloning vectors pMG103 and pMG105 were constructed and were stably maintained in R. palustris growing under nonselective conditions. The ability of plasmid pMG101 to replicate in R. palustris and its close phylogenetic relatives should enable broad application of these vectors within this group of alpha-proteobacteria.     

Mechanism of high-copy-number integration of pMIRY-type vectors into the ribosomal DNA of Saccharomyces cerevisiae

Targeted integration of the yeast plasmid pMIRY2 into the ribosomal DNA (rDNA) of Saccharomyces cerevisiae by homologous recombination results in transformants carrying 100-200 copies of the plasmid per cell which are stably maintained over a large number of generations [Lopes et al., Gene 79 (1989) 199-206]. These properties make pMIRY2 an attractive vector for high-level production of (heterologous) proteins by yeast cells. We have investigated the mechanism underlying high-copy-number (hcn) integration of pMIRY-type plasmids and show that either targeting to a location outside the rDNA locus or use of the wild-type LEU2, instead of the deficient LEU2d gene, as selection marker reduces the copy number to the low value characteristic of standard integrating (YIp-type) yeast plasmids. Further experiments demonstrate that the hcn of pMIRY-type plasmids is achieved by amplification of a small number of copies initially integrated into the rDNA locus. Amplification depends upon the strong selection pressure created by the extremely low expression of the deficient LEU2d gene, but not on the presence of this gene per se. The hcn integration also occurs when either the TRP1 or URA3 gene is used as the selection marker, provided expression of the marker gene is severely curtailed, e.g., by removal of most of its 5'-flanking region.     

Presence of nuclear associated plasmids in the lower eukaryote Dictyostelium discoideum

High copy number plasmids have been identified in six out of 25 wild-type strains of the cellular slime mould Dictyostelium discoideum, a model organism in developmental biology (Loomis, 1982). The characterization of three plasmids, from the NC4 (Ddp1), WS380B (Ddp2) and OHIO (Ddp3) wild isolates, is presented here. We show that they are nuclear associated and non-homologous to the mitochondrial DNA and extrachromosomal ribosomal DNA.