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.     

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.     

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.     

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.     

Dgp1 and Dfp1 are closely related plasmids in the Dictyostelium Ddp2 plasmid family

Dictyostelium plasmids Dgp1 and Dfp1, two members of the Ddp2 plasmid family, are 86% identical in nucleotide sequence. These small (4481 and 5015 bp), high copy number, nuclear plasmids carry both a gene homologous to the Ddp2 rep gene and a long 0.47- to 0. 48-kb inverted repeat region. Their Rep proteins are 82.8% identical in amino acid sequence and carry all 10 of the conserved peptide sequence motifs found in the Ddp2 family Rep proteins. Unlike other members of this family, Dgp1 carries two copies and Dfp1 carries four copies of a 162- to 166-bp direct repeat element. Both the direct and inverted repeat elements, as well as the promoter of the rep gene, are highly conserved (81 to 90% identical) between Dgp1 and Dfp1. In contrast, these regions are not highly conserved and the Rep proteins are only about 40% identical among the other known members of the plasmid family.     

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.     

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.     

Green fluorescent protein and epitope tag fusion vectors for Dictyostelium discoideum

We have constructed expression vectors for Dictyostelium discoideum which encode a green fluorescent protein (GFP) sequence upstream of a multicloning site for introduction of sequences of interest. Insertion of cDNAs into the multicloning site results in expression of fusion protein bearing an amino- or carboxyl-terminal GFP tag which can be used for fluorescent localization studies in Dictyostelium cells. A parallel construct fuses a FLAG epitope tag at the amino terminus of expressed protein. Each fusion cartridge was placed either in a G418-resistance vector allowing transactivated Ddp2-based extrachromosomal replication or in a vector allowing autonomous Ddp1-based replication. Distinct differences in expression stability were observed in the two vector types. When GFP-expressing cells were analyzed by fluorescence microscopy, significant cell-to-cell variability in expression level was observed when expression was based on the Ddp2 vector, while less cell-to-cell variation in expression level was observed when the Ddp1 backbone was used for expression.     

Identification of the origin of replication of the eukaryoteDictyostelium discoideum nuclear plasmid Ddp2

Ddp2 is a 5.8-kb, high-copy-number, nuclear plasmid found in the eukaryoteDictyostelium 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 inD. discoideum, provided that the Rep gene, which acts intrans, 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.     

Characterization of a retrovirus shuttle vector capable of either proviral integration or extrachromosomal replication in mouse cells.

A retrovirus shuttle vector is described that contains the dominant selectable neo gene which confers resistance to kanamycin in bacteria and to the drug G418 in animal cells. The bacterial supF gene and the origins of DNA replication from polyomavirus and the ColE1 replicon also have been included in this vector. Infection of normal rodent cells results in single-copy proviral integration, whereas infection of mouse (MOP) cells expressing polyoma large T antigen results in extrachromosomal replication of the DNA form of the virus. The copy number of the extrachromosomal circles in MOP cells varies from 0 to 100 copies per cell. G418-resistant MOP cells lose their drug-resistant phenotype after passage under nonselective conditions, suggesting that maintenance of the extrachromosomal circles is unstable. The extrachromosomal form of the virus can be recovered as plasmids in Escherichia coli. Two-thirds of the circles analyzed were found to be structurally intact. The others have undergone rearrangements including deletions and insertions. The bacterial supF gene was found to be intact in the majority of recovered plasmids. The data presented here suggest that these retroviruses should be useful as gene transfer vectors for animal cells in culture or in vivo.     

Copy number control and compatibility of nuclear plasmids in Dictyostelium discoideum

Copy number of the endogenous nuclear plasmids of Dictyostelium discoideum is a plasmid-specific trait. Copy number is stable over time, is constant relative to ploidy level, is independent of host cell genetic background, and is independent of the presence of a second unrelated plasmid in the same nucleus. Unrelated plasmids are compatible with one another within a single nucleus. Pairwise combinations of Ddp1, Ddp2, and Ddp5 were stably maintained over many generations in the absence of selection. In contrast, one of the D. discoideum plasmids (Ddp2) was incompatible with a recombinant plasmid derived from it (p7d2). In the absence of selection for retention of p7d2, transformants contain either one or the other but not both plasmids. The plasmids are stably maintained in host cells with differing genetic backgrounds, although plasmid-free colonies were detected at a frequency of about 1-2% in populations of some strains after 50 generations growth following a previous cloning.     

Compatible Dictyostelium mucoroides Nuclear Plasmids Dmp1 and Dmp2 Both Belong to the Ddp1 Plasmid Family

Dictyostelium mucoroides plasmids Dmp1 and Dmp2 are naturally occurring compatible members of the Dictyostelium Ddp1 plasmid family found in the same wild isolate strain. The nucleotide sequences of Dmp1 (5983 bp) and Dmp2 (6018 bp) are 74% identical and each carries open reading frames (ORFs) similar to the G1 and G5 ORFs of the Dictyostelium discoideum plasmid Ddp1. The predicted protein product of the Ddp1 G1 ORF is 49% similar to that of the Dmp1 G1-like ORF and 52% similar to that of the Dmp2 G1-like ORF. For the G5 and G5-like ORFs the corresponding values are 47 and 43%, respectively. The G1 and G5 ORFs of Ddp1 are transcribed during both vegetative growth and development of the asexual fruiting body. The G1-like ORF of Dmp2 is expressed in vegetative and developing cells, while that of Dmp1 appears to be expressed mostly in developing cells. The G5-like ORFs of Dmp1 and Dmp2 are expressed in both vegetative and developing cells. Dmp1 and Dmp2 differ from Ddp1 in (I) lacking homologs for the Ddp1 G2/G3/D4, G4/D5, D1/D3, and D2 ORFs; (2) containing multiple copies of a 173 bp direct repeat; and (3) having a different orientation of the G1 ORF relative to the G5 ORF. These findings suggest that the basic replicon unit of the Ddp1 plasmid family is composed of an origin of replication coupled to G1-like and G5-like genes. The additional ORFs and direct repeat elements in Ddp1, Dmp1, and Dmp2 may provide accessory functions beneficial to plasmid maintenance. Shuttle vectors based on Dmp1 or Dmp2 replicate in D. discoideum transformants.     

Extrachromosomal DNA isolated from tomato big bud and Candidatus Phytoplasma australiense phytoplasma strains

The nucleotide sequences of two extrachromosomal elements from tomato big bud (TBB) and one extrachromosomal element from Candidatus Phytoplasma australiense (Ca. P. australiense) phytoplasmas were determined. Both TBB plasmids (3319 and 4092 bp) contained an open reading frame ( approximately 570 bp) with homology to the rolling circle replication initiator protein (Rep). This gene was shorter than the rep genes identified from other phytoplasma plasmids, geminiviruses and bacterial plasmids. Both TBB extrachromosomal DNAs (eDNAs) encoded a putative DNA primase (dnaG) gene, a chromosomal gene required for DNA replication and which contains the conserved topoisomerase/primase domain. We speculate that the replication mechanism for the TBB phytoplasma eDNA involves the dnaG gene instead of the rep gene. The Ca. P. australiense eDNA (3773 bp) was shown to be circular and contained four open reading frames. The rep gene was encoded on ORF 1 and had homology to both plasmid (pLS1) and geminivirus-like domains.     

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.