Dgp1 and Dfp1 Are Closely Related Plasmids in theDictyosteliumDdp2 Plasmid Family

Plasmid 41, 89 –96 (1999) Article ID plas.1998.1385, available online at http://www.idealibrary.com on

Dgp1 and Dfp1 Are Closely Related Plasmids in the Dictyostelium Ddp2 Plasmid Family Chad M. Gonzales, Trina D. Spencer, Scott S. Pendley, and Dennis L. Welker 1 Program in Molecular Biology, Department of Biology, Utah State University, Logan, Utah 84322-5305 Received July 31, 1998; revised October 30, 1998 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. © 1999 Academic Press

repeat and a rep gene, as well as an additional five genes (Rieben et al., 1998). Sequences of four plasmids in the Ddp2 family (Ddp2, Ddp5, Ddp6, and pDG1) have been presented previously (Orii et al., 1989; Leiting et al., 1990; Slade et al., 1990; Rieben et al., 1998; Shammat et al., 1998). A major goal of the sequence analyses has been to identify conserved features within the plasmids. These may be important for plasmid maintenance and involved with cellular factors in initiating and controlling replication and transcription of the plasmid or they may be important structural elements, for example, in determining the three-dimensional shape of the Rep protein. Variation within the conserved regions may allow development of plasmid-specific features that are needed for the evolution of new independently replicating compatible plasmids such as Ddp2, Ddp5, and Ddp6, which can coexist, each at its own normal copy number, within a single cell (Hughes and Welker, 1989). The sequences of the previously described inverted repeat elements have been highly divergent and the Rep proteins of these plasmids are only about 40% identical in amino acid sequence. A set of 10 short conserved peptide sequence motifs has been identified in the Rep proteins (Leiting et al., 1990; Slade et

In Dictyostelium slime molds four families of nuclear plasmids, the Ddp1, Ddp2, Dpp1, and Dpp3 plasmid families, have been identified based on sequence and structural similarities (Orii et al., 1989; Leiting et al., 1990; Slade et al., 1990; Yin and Welker, 1992; Kiyosawa et al., 1993; Farrar et al., 1994; Kiyosawa et al., 1994; Rieben et al., 1998; Shammat et al., 1998). None of the Dictyostelium plasmids are similar to the nuclear plasmids found in yeast species (Volkert et al., 1989). Most work on Dictyostelium plasmids has focused on the Dictyostelium discoideum plasmids, since this species is used as a model system for cellular and developmental biology and since among the Dictyostelium species only D. discoideum is currently amenable to molecular genetic alterations of its genome through transformation. Plasmids in the Ddp2 plasmid family are circular, nuclear, high copy number DNA molecules which contain a long inverted repeat and a gene homologous to the Ddp2 rep gene (Orii et al., 1989; Leiting et al., 1990; Slade et al., 1990; Yin and Welker, 1992; Shammat et al., 1998). The sole exception to these observations is the Ddp5 plasmid which carries an inverted 1

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al., 1990; Shammat et al., 1998), but within these there are numerous differences among the plasmids. The high level of divergence of the plasmids made it difficult to identify features that could be reasonable targets for site-specific mutagenesis work aimed at understanding the molecular details of plasmid maintenance. In this report, we describe the sequence and structure of the Dgp1 plasmid from Dictyostelium giganteum, which is known to be a member of the Ddp2 plasmid family (Yin and Welker, 1992), and we identify and characterize a sixth member of the Ddp2 family, Dfp1, from Dictyostelium firmibasis. Analysis of the Dgp1 and Dfp1 sequences reveals that these plasmids are closely related. Not only are the rep genes of these two plasmids very similar but also the sequences of their repeat elements and gene promoters. Comparison of Dgp1 and Dfp1 with the other members of the plasmid family will help identify features of functional significance in plasmid maintenance. MATERIALS AND METHODS

and SP6 primers from multiple full-length cloned plasmid molecules, from deletion plasmids, and from plasmids derived by cloning smaller restriction fragments. The sequence surrounding all restriction sites used in the cloning was confirmed by overlapping sequence data. Further sequence data were obtained using specific primers. The sequencing was performed through the Utah State University Biotechnology Center using a Perkins-Elmer Applied Biosystems automated sequencer. Sequence data were compiled and analyzed using the GCG suite of DNA analysis programs with their default settings. The phylogenetic analysis of the Ddp2 plasmid family utilized the eprotdist and efitch programs from the WebANGIS collection of programs available from the Australian National Genomic Information Service. This analysis was based on a distance method employing the Kimura formula. The accession numbers for these and related sequences are Dgp1, U94491; Dfp1, AF076279; Ddp2, X51478 (but see also M55298); Ddp5, AF000580; Ddp6, U94410; and pDG1, X13703.

Strains and Plasmids

PCR Analysis

Dictyostelium wild isolate strains were screened for the presence of plasmids using a rapid miniscreen technique (Hughes and Welker, 1988). Plasmids from selected strains were isolated in large amounts, screened for the presence of unique restriction sites, and cloned into Escherichia coli vectors (Hughes et al., 1988; Hughes and Welker, 1989). The Dgp1 plasmid is found in D. giganteum wild isolate DG61 (also called D6-1 or 6.1) which was obtained from Dr. David Waddell (Yin and Welker, 1992). The Dfp1 plasmid is found in D. firmibasis isolate CR II 2B, which was obtained from Drs. John Landolt and David Francis. This strain was isolated from a soil sample from Costa Rica. Both D. giganteum and D. firmibasis are among the species closely related to Dictyostelium mucoroides.

To determine whether new plasmids were members of the Ddp2 plasmid family they were screened by a PCR-based test. A pair of degenerate primers specific for the nucleotide sequences corresponding to parts of the Box 2 and Box 5 conserved peptide motifs in the Rep proteins of the Ddp2, Ddp5, Ddp6, Dgp1, and pDG1 plasmids was used. The primers were GGGTCTAGAARTSWGAWARRGTHRTWAC and GAAATYTCTKRRCGRCARTG, respectively (where H 5 A, C, or T; K 5 G or T; R 5 A or G; S 5 C or G; W 5 A or T; and Y 5 C or T). In Ddp2 the primers paired with the nucleotides at positions 3473 to 3492 and 4139 to 4158. Amplification by PCR yields products ranging from 679 to 700 bp on the different members of the Ddp2 plasmid family. RESULTS

Sequence Analysis Unambiguous data for the entire sequence for each plasmid were obtained using universal T7

The D. giganteum plasmid Dgp1 is a circular, high copy number, nuclear plasmid carrying a rep gene homolog and an inverted repeat (Yin

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Dictyostelium PLASMIDS Dgp1 AND Dfp1

FIG. 1. Structural maps of the Ddp2 plasmid family. The circular plasmids are shown linearized at the start codons of their rep genes. Smaller arrows indicate the positions of the 0.16-kb direct repeats in Dfp1 and Dgp1. Larger arrows indicate the positions of the long inverted repeats, some of which overlap much of the rep gene promoter, in particular in Ddp5. The dots in the center of the Ddp5 map indicate the location of the five additional genes present in Ddp5, these are homologous to genes on Ddp1 (Rieben et al., 1998). The sizes of the plasmid genes and inverted repeat elements are given in Table 1.

and Welker, 1992). The complete Dgp1 sequence, obtained in this work, contains 4481 bp with a G1C content of 24.2%, typical for Dictyostelium DNA. A pair of 0.48-kb inverted repeat elements separated by a short unique sequence region of 106 bp lies upstream of the promoter of the Dgp1 rep gene (Fig. 1, Table 1). The rep orf contains 2616 bp followed by UAA, which is the typical stop codon for Dictyostelium. The Dgp1 Rep protein has a length and structure similar to those of other members of the Ddp2 plasmid family (Fig. 2). The Dgp1 Rep protein carries all 10 of the previously identified conserved peptide motifs. It is the smallest of the Rep proteins, primarily because it has the shortest acidic region at its carboxy

terminus. Dgp1 appears to be about equally related to the four previously described Ddp2 family members (pDG1, Ddp2, Ddp5, Ddp6), based on comparisons of the Rep protein sequences (Fig. 3). In a screen for new Dictyostelium plasmids, we identified the 5.0-kb Dfp1 plasmid in the D. firmibasis wild isolate CR II 2B. Dfp1 was cloned at its unique EcoRI site and used as a template in a PCR-based screen to determine whether it was a member of the Ddp2 plasmid family. Degenerate primers for the screen were prepared based on the sequences of two conserved regions in the rep genes of the known Ddp2 family members and allowed amplification of an 0.7-kb fragment from Dfp1, as well as from the known members of the Ddp2 plasmid family. Three other newly identified Dictyostelium plasmids when handled similarly did not yield a 0.7-kb fragment, indicating that they are not likely to be members of the Ddp2 plasmid family. Sequencing of Dfp1 revealed a length of 5015 bp, with a G1C content of 23.9%. There is a single long 2622-bp orf, terminating at a UAA stop codon, with homology to the Ddp2 rep gene. The Dfp1 Rep protein contains all 10 of the conserved peptide motifs seen in the other Ddp2 family Rep proteins (Fig. 2). Upstream of the promoter of this gene lies a long inverted repeat with a pair of 0.47-kb repeat units separated by a short unique sequence of 113 bp (Fig. 1).

TABLE 1 Dictyostelium Ddp2 Plasmid Family Characteristics

Plasmid

Size (bp)

rep orf (bp)

Rep protein (amino acids)

Inverted repeat elements (bp)

Short unique sequence (bp)

Ref. a

Dfp1 Dgp1 Ddp2 Ddp5 Ddp6 pDG1

5015 4481 5852 14955 5257 4439

2622 2616 2661 3114 2796 2718

874 872 887 1038 932 906

475, 471 478, 475 501, 501 689, 705 656, 655 552, 551

113 106 65 225 70 80

1 1 2 3 4 5

Note. The short unique sequence is the region separating the two inverted repeat elements. Ddp5 is bigger than the other Ddp2 plasmid family members because it carries five additional genes. a References: 1, this work; 2, Slade et al., 1990; 3, Rieben et al., 1998; 4, Shammat et al., 1998; 5, Orii et al., 1989.

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FIG. 2. The Rep proteins of the Ddp2 plasmid family. Bold lettering indicates the position of 10 conserved peptide sequence motifs found in other members of the Ddp2 plasmid family (Shammat et al., 1998). These motifs may have structural or functional significance. The numbering is a relative scale for the aligned peptides. Amino acids conserved in all six peptides are indicated with an asterisk under the aligned sequence.

Dictyostelium PLASMIDS Dgp1 AND Dfp1

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FIG. 2—Continued

Comparison of the Dfp1 sequence with those of the other Ddp2 family plasmids revealed that Dfp1 is most closely related to the Dgp1 plasmid with 86% identity at the nucleotide level. The predicted Dfp1 and Dgp1 Rep proteins are 89.8% similar and 82.8% identical. The Dfp1 and Dgp1 Rep proteins are more distantly related to the Rep proteins of the other Ddp2 family members (Fig. 2 and Fig. 3), being about

60% similar and 40% identical. The amino acids within the polythreonine box and the acidic carboxy terminus are identical in Dfp1 and Dgp1, and in the 8 other conserved motifs only 4 of 84 residues are different, three of these substitutions are conservative changes. In contrast, within these 84 residues even plasmids from the same Dictyostelium species typically have numerous plasmid-specific changes. For

FIG. 3. Radial “phylogenetic” tree of the Ddp2 plasmid family based on the amino acid sequences of the Rep proteins. The tree shows that the Dgp1 and Dfp1 proteins are the most similar and that the Ddp5 protein is the most dissimilar of these proteins.

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example, 18 of the 84 residues differ between the Ddp2 and Ddp6 Rep proteins. The greatest differences between the Dfp1 and Dgp1 Rep proteins lie in three regions, one between amino acid residues 178 to 206, one surrounding the polythreonine box, and the other just upstream of the acidic carboxy terminus (Fig. 2). The sequence conservation between Dfp1 and Dgp1 extends outside the rep gene. The 0.43-kb region immediately following the Dfp1 rep gene (position 2626 to 3052) is one of the more divergent regions, with an additional 0.17 kb compared to the corresponding region of Dgp1 and with only 57% identity to the Dgp1 sequence. Following this divergent region is a direct repeat region with 162 to 166 bp per repeat. Dfp1 has four copies of this repeat and Dgp1 has two copies. These repeats are 90% identical. Following this region are the inverted repeats, which are 87% identical. In both plasmids the inverted repeat region spans a total of 1059 bp with the 0.47- to 0.48-kb repeat elements being separated by a unique sequence region of 113 bp in Dfp1 and 106 bp in Dgp1. These short unique regions are more divergent than the inverted repeat elements, being only 54% identical. Following the inverted repeats are the promoters and 59 transcribed leaders upstream of the start codons for the rep genes. The promoters of both Dfp1 and Dgp1 are rich in poly(T)/poly(A) tracts and are 81% identical. About 80 to 100 bp upstream of the start codons for the Dgp1 and Dfp1 rep genes are conserved CA-rich sequence elements. This feature is shared with the promoters of the rep genes in other Ddp2 family members, particularly Ddp2, Ddp6, and pDG1. Close to the start codons the Dfp1 and Dgp1 sequences again diverge. Dgp1 has three copies of the short direct repeat TTTTCATA and Dfp1 has four copies and part of a fifth copy of a CAAATAAAAATA repeat. DISCUSSION Dgp1 and Dfp1 are both members of the Ddp2 plasmid family, the most widely distributed Dictyostelium plasmid family. With this report, plasmids in this family have been identified in four different species recovered from

North America, Central America, and Japan. Dgp1 and Dfp1 share two features conserved in the Ddp2 plasmid family, a long inverted repeat and a gene homologous to the Ddp2 rep gene (Table 1, Fig. 1). They each contain a long direct repeat of 162 to 166 bp not found in the other Ddp2 plasmid family members. Dgp1 and Dfp1 are the most closely related members of the Ddp2 plasmid family (Fig. 3), being 86% identical at the nucleotide level. The other plasmid family members are only about 50% identical at the nucleotide level. The similarity of Dgp1 and Dfp1 exists not only in their rep genes but throughout the remainder of the plasmids including their inverted repeats, direct repeats, and rep gene promoters, which are 81 to 90% identical. The distribution of the Ddp2 plasmid family among several Dictyostelium species and the divergence in the plasmid sequences suggest that the plasmid family originated in a common ancestor of the current species. Structural relationships of the Ddp2 Rep protein to viral proteins have been described (Leiting et al., 1990) and may indicate evolution from a viral ancestor. The close relationship of Dgp1 and Dfp1 may reflect recent transfer between species. Interspecific transfer of native Dictyostelium plasmids, while typically unsuccessful, has been accomplished by transformation in our laboratory (Hughes et al., 1988). The Ddp2 replication origin appears to lie in the region overlapping the part of the inverted repeat and neighboring region just upstream of the rep gene promoter (Leiting et al., 1990; Chang et al., 1990; Hughes et al., 1992). Binding of the Rep protein in this region or adjacent to it may control both origin function and transcription (Leiting et al., 1990; Chang et al., 1990; Slade et al., 1990; Shammat et al., 1998). Each independently replicating plasmid population requires the ability to form plasmid-specific protein and DNA complexes, in particular to control plasmid replication and copy number. Coevolution of the sequence of a DNA site on the plasmid and of the DNA-binding domain in the Rep protein as plasmids diverge would allow independently replicating populations of compatible plasmid types to be established

Dictyostelium PLASMIDS Dgp1 AND Dfp1

(Hughes and Welker, 1989; Kiyosawa et al., 1994). Two regions of interest as potential protein-binding sites within the Dgp1 and Dfp1 promoters are the short direct repeats just upstream of the start codons of the rep genes and the CA-rich elements found 80 to 100 bp upstream of the start codon. Neither of the corresponding regions was absolutely essential for replication of Ddp2-based vectors (Leiting et al., 1990; Chang et al., 1990; Hughes et al., 1992), but the Ddp6 Rep protein was shown to position nucleosomes in the Ddp6 promoter region (Shammat, 1997; Shammat and Welker, unpublished results), suggesting that it binds to the Ddp6 promoter region. Within the short direct repeats are multiples of 7-bp sequences, ATACAAA in Dfp1 and TTTCATA in Dgp1. The promoters of other Ddp2 family members also contain related sequences, ATTCAAA in Ddp2 and Ddp6, TTTCGAT in Ddp5, and TTACAAA in pDG1. The presence of the CArich sequence elements in the Ddp2 and pDG1 plasmids and their potential role in promoter function have been noted previously but not further studied (Slade et al., 1990). The presence of CA-rich elements at similar locations in all members of the plasmid family strongly suggests a role for this sequence element in control of rep gene expression. The tetranucleotide CACA is also found in four locations in each of the Dgp1 and Dfp1 inverted repeat elements. The role in plasmid maintenance of the inverted repeat and of the region between the 39 end of the rep gene and the inverted repeat also requires further characterization. It is clear that in Ddp2 and Ddp6 these regions are involved in plasmid maintenance (Hughes et al., 1992; Shammat et al., 1998), but the mechanism(s) used is unknown. Although the direct repeats shared by Dgp1 and Dfp1 lie in this region, the Dgp1 and Dfp1 plasmid sequences are very divergent from the other members of the plasmid family making it difficult to identify potentially significant features. Long direct repeats have been identified in other Dictyostelium plasmids in the Ddp1 and Dpp3 plasmid families, but their functional significance is not known (Kiyosawa et al., 1993, 1994; Rieben et al., 1998). In yeast plasmids direct repeats play

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a role in plasmid partitioning. Comparison of the Dgp1 and Dfp1 inverted repeat elements reveals several interesting features. There are positions in which both copies of the Dfp1 repeat elements are identical but different from those of Dgp1. This implies that concerted evolution of the repeats is occurring, which may be important for development of plasmid-specific sequences. Almost all of this type of change occurs in the 0.3 kb of the repeat elements that lie adjacent to the central unique sequence region. Outside of this region, in the remaining 0.17 kb of the repeat elements, sequence variation also exists. Much of this variation is between elements within the same plasmid and is conserved in Dgp1 and Dfp1, which suggests that specialization of the function of the two inverted repeat elements may be important for plasmid maintenance. The repeat element nearest the 59 end of the rep gene may be specialized for normal origin function. This is consistent with earlier work in which the Ddp2 replication origin was localized to this region (Leiting et al., 1990; Chang et al., 1990). The repeat element nearest the 39 end of the rep gene may be involved in plasmid partitioning or copy number amplification. The Ddp2 and Ddp6 Rep proteins are known to be critical for maintenance of plasmid-based shuttle vectors under nonselective growth conditions (Hughes et al., 1992; Shammat et al., 1998). Evidence from other work in our laboratory indicates that Rep proteins can form plasmid-specific protein mutimers and bind plasmid DNA (Shammat and Welker, unpublished data; Shammat, 1997). The Rep proteins of Dgp1 and Dfp1 are 82.8% identical. The differences are scattered throughout the protein with three regions having a higher degree of variation (Fig. 2). Plasmid-specific sequence variation is expected in the protein’s DNA-binding domain and multimerization domain. However, it is anticipated that these domains would have a higher than average level of conservation from one Rep protein to another, reflecting retention of specific residues at important sites. None of the three most divergent regions between the Dfp1 and Dgp1 proteins seem to fit this criterium. It is likely that the regions of the Rep

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protein with plasmid-specific functions are those with an intermediate level of sequence divergence in Dgp1 and Dfp1. The small amount of variation in the conserved peptide sequence motifs, where only 4 of 84 residues differ in Dgp1 versus Dfp1, suggests that some of these peptide regions are involved in determining Rep protein structure or in the interaction of Rep protein with cellular replication and/or transcription factors. Others, such as the Box 2 and Box 6 motifs, lie in regions with some sequence differences (Fig. 2) and are more likely to have plasmid-specific functions. ACKNOWLEDGMENTS We thank our colleagues Drs. D. Waddell, J. Landolt, and D. Francis for their generous gift of strains. We also thank the reviewers and editor for their suggestions. C.M.G. and T.D.S. were supported by undergraduate research fellowships under the auspices of a grant from the Howard Hughes Medical Institute to the USU Biology Department. This work was supported by funds from USU.

REFERENCES Chang, A. C. M., Slade, M. B., and Williams, K. L. (1990). Identification of the origin of replication of the eukaryote Dictyostelium discoideum nuclear plasmid Ddp2. Plasmid 24, 208 –217. Farrar, N. A., Kiyosawa, H., Hughes, J. E., Welker, D. L., and Williams, K. L. (1994). Nucleotide sequence of Ddp1, a high copy number nuclear plasmid of Dictyostelium discoideum. Plasmid 31, 184 –195. Hughes, J. E., Ashktorab, H., and Welker, D. L. (1988). Nuclear plasmids in the Dictyostelium slime molds. Dev. Genet. 9, 495–504. Hughes, J. E., Podgorski, G. J., and Welker, D. L. (1992). Selection of Dictyostelium discoideum transformants and analysis of vector maintenance using live bacteria resistant to G418. Plasmid 28, 48 – 60. Hughes, J. E., and Welker, D. L. (1988). A mini-screen technique for analyzing nuclear DNA from a single Dictyostelium colony. Nucleic Acids Res. 16, 2338.

Hughes, J. E., and Welker, D. L. (1989). Copy number control and compatibility of nuclear plasmids in Dictyostelium discoideum. Plasmid 22, 215–223. Kiyosawa, H., Hughes, J. E., Podgorski, G. J., and Welker, D. L. (1993). Small circular plasmids of the eukaryote Dictyostelium purpureum define two novel plasmid families. Plasmid 30, 106 –118. Kiyosawa, H., Hughes, J. E., and Welker, D. L. (1994). Compatible Dictyostelium mucoroides nuclear plasmids Dmp1 and Dmp2 both belong to the Ddp1 plasmid family. Plasmid 31, 121–130. Leiting, B., Lindner, I. J., and Noegel, A. A. (1990). The extrachromosomal replication of Dictyostelium discoideum plasmid Ddp2 requires a cis-acting element and a plasmid-encoded trans-acting factor. Mol. Cell. Biol. 7, 3727–3736. Orii, H., Tanaka, Y., and Yanagisawa, K. (1989). Sequence organization and gene expression of pDG1, a plasmid found in a wild isolate of Dictyostelium. Nucleic Acids Res. 17, 1395–1408. Rieben, W. K., Gonzales, C. M., Gonzales, S. T., Pilkington, K. J., Kiyosawa, H., Hughes, J. E., and Welker, D. L. (1998). Dictyostelium discoideum nuclear plasmid Ddp5 is a chimera related to the Ddp1 and Ddp2 plasmid families. Genetics 148, 1117–1125. Shammat, I. (1997). Studies of Dictyostelium discoideum plasmid Ddp6 and on the mechanism of action of Rep proteins from the Ddp2 plasmid family. Ph.D. Dissertation. Utah State University, Logan, UT. Shammat, I. M., Gonzales, C. M., and Welker, D. L. (1998). Dictyostelium discoideum nuclear plasmid Ddp6 is a new member of the Ddp2 plasmid family. Curr. Genet. 33, 77– 82. Slade, M. B., Chang, A. C. M., and Williams, K. L. (1990). The sequence and organization of Ddp2, a high-copynumber nuclear plasmid of Dictyostelium discoideum. Plasmid 24, 195–207. Volkert, F. C., Wilson, D. W., and Broach, J. R. (1989). Deoxyribonucleic acid plasmids in yeast. Microbiol. Rev. 53, 299 –317. Yin, Y., and Welker, D. L. (1992). Dictyostelium giganteum plasmid Dgp1 is a member of the Ddp2 plasmid family. Plasmid 28, 37– 45. Communicated by J. I. Rood