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.     

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.     

Cloning and sequence analysis of a plasmid replicon from Lactococcus lactis subsp. cremoris FG2

A replication region from one of the Lactococcus lactis subsp. cremoris FG2 plasmids was isolated by cloning of a 4.8-kb XbaI fragment into a replication probe vector and transformation into L. lactis LM0230. A 1.8-kb region within this fragment was sequenced and confirmed by PCR subcloning to encode a functional replicon in LM0230. The replicon consists of an open reading frame encoding a putative replication protein (Rep) of 386 amino acids and a non-coding region (ori) which features several structural motifs typical of other known replication origins, including a 22-bp iteron sequence tandemly repeated three and a half times, a 10-bp direct repeat and two sets of inverted repeats. The ori region could drive replication of its plasmid when supplied with the replication region in-trans. The lack of detectable single-stranded DNA during replication and the existence of extensive homology with other known lactococcal theta replicons strongly suggest that this region encodes a theta-replicating mechanism.     

Relationship among location of T-antigen-induced DNA distortion,auxiliary sequences,and DNA replication efficiency

T-antigen-induced DNA distortion was studied in a series of simian virus 40 (SV40) plasmid constructs whose relative replication efficiency ranges from 0.2 to 36. Bending was detected in the wild-type SV40 regulatory region consisting of three copies of the GC-rich 21-bp repeat but not in constructs with only one or two copies of the 21-bp repeat. In a construct with enhanced replication efficiency, bending occurred in a 69-bp cellular sequence located upstream of a single copy of the 21-bp repeat. Bending occurred both upstream of ori and in the three 21-bp repeats located downstream of ori in a construct with reduced replication efficiency. In a construct with no 21-bp repeats, DNA distortion occurred downstream of ori. The results indicate that SV40 DNA replication is enhanced when the structure of the regulatory region allows the DNA to form a bent structure upstream of the initial movement of the replication fork.     

Molecular characterization of the minimal replicon and the unidirectional theta replication of pSCM201 in extremely halophilic archaea

A 3,463-bp plasmid, pSCM201, was isolated from a halophilic archaeon, Haloarcula sp. strain AS7094. The minimal replicon that is essential and sufficient for autonomous replication and stable maintenance in Haloarcula hispanica was determined by deletion analysis of the plasmid. This minimal replicon ( approximately 1.8 kb) consisted of only two functionally related segments: (i) a putative origin (ori201) containing an AT-rich region and sets of repeats and (ii) an adjacent gene encoding a putative replication initiation protein (Rep201). Electron microscopic observation and Southern blotting analysis demonstrated that pSCM201 replicates via a theta mechanism. Precise mapping of the putative origin suggested that the replication initiated from a fixed site close to the AT-rich region and proceeded unidirectionally toward the downstream rep201 gene, which was further confirmed by electron microscopic analysis of the ClaI-digested replication intermediates. To our knowledge, this is the first unidirectional theta replication plasmid experimentally identified in the domain of archaea. It provides a novel plasmid system to conduct research on archaeal DNA replication.     

Simian virus 40 DNA replication: functional organization of regulatory elements.

The efficiency of simian virus 40 (SV40) DNA replication is dependent on the structural organization of the regulatory region. The enhancing effect of the G + C-rich 21-base-pair (bp) repeats on SV40 DNA replication is position and dose dependent and to some extent orientation dependent. The inverted orientation is about 50% as effective as the normal orientation of the 21-bp repeat region. Movement of the 21-bp repeat region 180 or 370 bp upstream of the ori sequence abolishes its enhancing effect, whereas no replication is detected if the 21-bp repeat region is placed downstream of the ori sequence. The dose-dependent enhancement of the 21-bp repeat of SV40 DNA replication as first described in single transfection by Bergsma et al. (D. J. Bergsma, D. M. Olive, S. W. Hartzell, and K. N. Subramanian, Proc. Natl. Acad. Sci. USA 79:381-385, 1982) is dramatically amplified in mixed transfection. In the presence of the 21-bp repeat region, the 72-bp repeat region can enhance SV40 DNA replication. In the presence of the 21-bp repeats and a competitive environment, the 72-bp repeat region exhibits a cis-acting inhibitory effect on SV40 DNA replication.     

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.     

DNA sequence and analysis of a cryptic 4.2-kb plasmid from the filamentous cyanobacterium, Plectonema sp. strain PCC 6402.

The 4194-bp plasmid, pRF1, from Plectonema sp. Strain PCC 6402 was completely sequenced and analyzed. Seven potential open reading frames were identified. The predicted amino acid sequence of open reading frame C (ORF C) had identities of 34, 29, and 25% with Rep B from the Staphylococcus aureus plasmid, pUB110; Rep from the Bacillus amyloliquefaciens plasmid, pFTB14; and protein A from the S. aureus plasmid, pC194, respectively. A 75-amino-acid region conserved in these proteins (Rep B, Rep, and protein A) also was highly conserved in ORF C with identities of 45, 37, and 40%, respectively. Significantly, 16 of the 21 amino acids conserved in Rep B, Rep, and protein A were found at the same positions in ORF C. This ORF may encode a replication protein that includes a region conserved in some eubacteria. Additional structural features include a 425-bp region that contains palindromes, tandem repeats, and short direct repeats which may correspond to the origin of replication. An 18-bp inverted repeat was located between two open reading frames, A and G.     

The replication origin of pSC101: the nucleotide sequence and replication functions of the ori region

The nucleotide sequence of a 770-bp ori region of plasmid pSC101 is presented. The sequence shows homologies to some parts of Escherichia coli oriC and phage G4 ori. Several other features are an 80-bp A + T-rich region overlapping a part of the region homologous to oriC, three direct repeats of an 18-bp sequence adjacent to the A + T-rich region, a typical promoter sequence just upstream of the longest open reading frame (ORF) and a long inverted repeat sequence overlapping the putative promoter region. Analysis of successive deletions by BAL31 exonuclease demonstrated that one of the regions homologous to oriC along with the A + T-rich region are essential for autonomous replication of the plasmid. The three 18-bp repeats are responsible for incompatibility phenotype. The region containing the promoter-like sequence is required for expression of a trans-acting function.     

Cloning and sequencing of plasmid pLC494 isolated from human intestinal Lactobacillus casei: construction of an Escherichia coli-Lactobacillus shuttle vector

The complete nucleotide sequence of plasmid pLC494 isolated from Lactobacillus casei L-49 was determined. Plasmid pLC494 is an 8846-bp long circular molecule with a G+C content of 41.5%. Two putative open reading frames, ORF4 (282 amino acids) and ORF5 (169 amino acids), were identified as replication proteins A and B that revealed 100 and 99% similarity, respectively, with the replication proteins of plasmid pLA103 from Lactobacillus acidophilus TK8912. Upstream of ORF4 were the four repeat regions (three perfect 22-bp repeats and one imperfect motif), a putative ribosome binding site, a -10 region, and a -35 region. The shuttle vector pJLE4942 (5318 bp) was constructed using repA from pLC494, a multiple cloning site, ColE1 ori, the ori of gram-negative bacteria from vector pUC19, and the chloramphenicol resistance gene from pJIR418 as a selection marker. Transformation of several lactic acid bacteria with the vector pJLE4942 indicated that this vector might be useful as a genetic tool for the intestinal lactobacilli.     

pHP489, a Helicobacter pylori small cryptic plasmid, harbors a novel gene coding for a replication initiation protein

We have analyzed a Helicobacter pylori plasmid, pHP489. The 1222-bp nucleotide sequence had one open reading frame, a DnaA-binding site, one direct repeat, and three inverted repeats. The (G+C) content of pHP489 was 33.3%. Although the nucleic acid sequence and deduced amino acid sequence were homologous to those of other bacterial plasmid Rep proteins, the degree of similarity was very low. A deletion analysis showed that the Rep protein was not required for the replication of pHP489 in its H. pylori host, but the host replication machinery was needed.     

Exonuclease III promotes in vitro binding of the replication initiator protein of plasmid pSC101 to the repeated sequences in the ori region

Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S. et al. (1990) J. Biochem. 107, 369-376). We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli. In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep. On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor. We purified extensively the factor and identified it as exonuclease III (exo III). Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA. This binding of Rep to duplex DNA treated with exo III is DR-sequence specific. Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.     

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.     

Two enhancer elements for DNA replication of pSC101, par and a palindromic binding sequence of the Rep protein.

The minimal replication origin (ori) of the plasmid pSC101 has been previously defined as an approximately 220-bp region by using plasmids defective in the par region, which is a cis-acting determinant of plasmid stability. This ori region contains the DnaA binding sequence, three repeated sequences (iterons), and an inverted repeat (IR) element (IR-1), one of the binding sites of an initiator protein, Rep (or RepA). In the present study, we show that plasmids containing par can replicate at a nearly normal copy number in the absence of IR-1 but still require a region (the downstream region) between the third iteron and IR-1. Because par is dispensable in plasmids retaining IR-1, par and IR-1 can compensate each other for efficient replication. The region from the DnaA box to the downstream region can support DNA replication at a reduced frequency, and it is designated "core-ori." Addition of either IR-1 or par to core-ori increases the copy number of the plasmid up to a nearly normal level. However, the IR-1 element must be located downstream of the third iteron (or upstream of the rep gene) to enhance replication of the plasmid, while the par region, to which DNA gyrase can bind, functions optimally regardless of its location. Furthermore, the enhancer activity of IR-1 is dependent on the helical phase of the DNA double helix, suggesting that the Rep protein bound to IR-1 stimulates the activation of ori via its interaction with another factor or factors capable of binding to individual loci within ori.     

Minimal essential origin of plasmid pSC101 replication: requirement of a region downstream of iterons.

The minimal replication origin (ori) of the plasmid pSC101 was defined as an about 220-bp region under the condition that the Rep (or RepA) protein, a plasmid-encoded initiator protein, was supplied in trans. The DnaA box is located at one end of ori, as in other plasmids, like mini-F and P1. The other border is a strong binding site (IR-1) of Rep which is palindromic sequence and lies in an about 50-bp region beyond the repeated sequences (iterons) in ori. This IR-1 is located just upstream of another strong Rep binding site (IR-2), the operator site of the structure gene of Rep (rep), but its function has not been determined. The present study shows that the IR-1 sequence capable of binding to Rep is essential for plasmid replication with a nearly normal copy number. Furthermore, a region between the third iteron and IR-1 is also required in a sequence-specific fashion, since some one-base substitution in this region inactivate the origin function. It is likely that the region also is a recognition site of an unknown protein. Three copy number mutations of rep can suppress any one-base substitution mutation. On the other hand, the sequence of a spacer region between the second and the third iterons, which is similar to that of the downstream region of the third iteron, can be changed without loss of the origin function. The requirement of the region downstream of iterons in pSC101 seems to be unique among iteron-driven plasmid replicons.