*Department of Biological Sciences, University of Iowa;
and
Department of Botany, Canadian Institute for Advanced Research, University of British Columbia, Vancouver, British Columbia, Canada
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Abstract |
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Introduction |
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An issue of which nothing is presently known is the fate of nucleomorph genes that encode proteins with functions not required in the plastid or the PC. These sequences are presumably rapidly eliminated, but it is also conceivable that some may find their way to the host nuclear genome through gene transfer. This would be analogous to the process of endosymbiotic gene replacement that now appears to be common with prokaryotic endosymbiotic organelles such as the mitochondrion and the plastid (Keeling and Doolittle 1997
; Martin and Herrmann 1998
). Our study reports for the first time such a finding for the cryptophyte Pyrenomonas helgolandii. In this taxon, an actin gene from the red algal secondary symbiont has been transferred to the host nucleus, where it is expressed and apparently has assumed a novel function. This process is essentially a lateral gene transfer from one eukaryote to a distantly related eukaryote and provides a new source of genetic diversity that is potentially very important in eukaryotic evolution.
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Materials and Methods |
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Southern Blot Analyses
Genomic DNA was isolated from a P. helgolandii culture using the Plant DNeasy mini kit (Qiagen) and digested to completion with the BamHI and SacI restriction endonucleases under standard conditions (New England Biolabs). The digested DNA was transferred onto a nylon membrane (Schleicher and Schuell) and probed with partial cDNA fragments of the highly divergent and cryptophyte-like genes that had been isolated using gene-specific primers (underlined sequence in fig. 1
) and 5' RACE (see fragments in fig. 6A
). Another Southern blot was probed with an 1,100-bp cDNA fragment that encoded the nearly complete cryptophyte-like actin gene (isolated using primers Ac1 and Ac3). The Southern hybridizations were performed using a nonradioactive method according to the manufacturer's instructions (Gene Images, Amersham). The hybridization was done in 5 x SSC, 0.1% (w/v) SDS, and 5% (w/v) dextran sulphate at 60°C overnight. Filters were initially washed for 15 min at 60°C in 1 x SSC, and 0.1% SDS and then for 15 min at the same temperature in 0.1 x SSC and 0.1% SDS prior to detection. The same method was used to analyze a Southern blot of a pulsed-field gel containing DNA from the nuclear and nucleomorph compartments of G. theta (kindly provided by U.-G. Maier, Marburg, Germany). The divergent actin sequence was used as the probe in this experiment.
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Results and Discussion |
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Evidence that the P. helgolandii Red Algalike Actin Is Encoded in the Host Genome and Functions in the Host Cytoplasm
The presence of a red algalike actin gene in P. helgolandii could be explained in three ways. First, the sequence is encoded on the P. helgolandii nucleomorph genome, and its product is a cytoskeletal element in the PC. Recently, -, ß-, and
-tubulin genes have been found in the nucleomorph genome of the cryptomonad G. theta (Keeling et al. 1999
). Therefore, even though electron microscopic studies fail to detect any cytoskeletal elements such as actin, tubulin, or intermediate filaments in the PC of cryptomonads or chlorarachniophytes (Gillott and Gibbs 1980
; McKerracher and Gibbs 1982
; Morrall and Greenwood 1982
; Ludwig and Gibbs 1989
), at least tubulin-based cytoskeletal structures must still exist in the endosymbiont (Keeling et al. 1999
). Second, the gene has been transferred from the endosymbiont genome to the host nuclear genome of P. helgolandii and is posttranslationally imported back to the endosymbiont cytoplasm much as plastid proteins are targeted. Third, the gene has been transferred from the endosymbiont genome to the host nuclear genome and now functions as a novel actin in the host cytoplasm.
The first alternative was excluded by determining whether the red algalike gene was encoded in the nuclear or the nucleomorph genome. A heterologous Southern blot analysis was done with total DNA from the well-studied cryptomonad G. theta. The DNA in the different genetic compartments of G. theta was separated by pulsed-field gel electrophoresis (Hofmann et al. 1994
), and the resulting blot was probed with the P. helgolandii red algalike gene. This analysis showed a strong hybridization of the red algalike gene probe with the nuclear genome, but no hybridization with the three nucleomorph chromosomes (fig. 5
). This suggests that no actin-like sequence is encoded by the G. theta nucleomorph genome, and it provides circumstantial evidence that the red algalike gene is more likely encoded in the P. helgolandii host nucleus. However, RT-PCR analyses with G. theta total RNA and a 5' primer specific to the 5'-terminus of the P. helgolandii red algalike gene failed to amplify an ortholog of the red algalike actin gene in G. theta. If the red algalike actin gene in P. helgolandii is still being encoded in the nucleomorph genome, then the hybridization results from G. theta must be due to either differential transfer of nucleomorph genes in P. helgolandii and G. theta, the high divergence of the 5'-terminus of this gene, or the loss of this gene in G. theta. Pulsed-field gel analysis of total genomic DNA from P. helgolandii is needed to conclusively distinguish between these hypotheses.
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To address the possibility that the red algalike actin is posttranslationally targeted to the endosymbiont, we used 5' RACE procedures to complete the 5'-terminal sequences of the red algalike and cryptophyte-like actin cDNAs. Proteins targeted to the secondary endosymbiotic plastid of cryptophytes are first directed to the endomembrane system using an amino-terminal signal peptide (McFadden 1999
; Wastl and Maier 2000
). It is likely that any protein targeted to the endosymbiont cytosol will initially use the same protein trafficking pathway, so if this actin is targeted to the endosymbiont, it is predicted to encode a signal peptide. The prediction of an "extra" sequence at the 5'-terminus of the red algalike gene was supported with the finding of a 5' RACE product that was of a relatively larger size than the cryptophyte-like cDNA (fig. 6A
). However, sequence analyses of these cloned PCR products provided an unexpected result. The extra sequence at the 5'-terminus of the red algalike gene did not share any properties with cryptophyte signal sequences (e.g., Durnford et al. 1999
; McFadden 1999
; Wastl et al. 1999, 2000
; Wastl and Maier 2000
) but, rather, was a highly hydrophilic, novel domain not yet described for actins or any other protein (fig. 6B
). Although the N-terminus is the most variable region of actins, this domain never shows an additional sequence, as is apparent in the red algalike actin (e.g., see alignment in Sheterline, Clayton, and Sparrow 1995
). This is significant because the variable N-terminus is thought to facilitate the binding of actin to a variety of different actin-binding proteins (ABPs; Herman 1993
; Bhattacharya and Ehlting 1995
; Sheterline, Clayton, and Sparrow 1995
). The different ABPs confer the diversity of functions ascribed to this otherwise highly conserved protein. With this in mind, we postulate that the red algalike actin has evolved a new actin-related function in the host cell through the evolution of a novel, divergent N-terminal domain. Analysis of the amino acid sequence in the domain suggests that it originated through the duplication of an SDEE amino acid motif (fig. 6B
) separated by a glycine (G) residue. That the serine (S) residues are encoded by the same codon (TCC; out of six possible triplets) in both repeats is consistent with a recent origin of this sequence through an internal duplication. The preponderance of charged amino acids such as aspartic acid (D) and glutamic acid (E) suggests that the novel, likely
-helical, domain is completely exposed in the red algalike actin tertiary structure and interacts with another protein (Branden and Tooze 1991
). Although we propose a host function for the red algalike actin, it is also possible but unlikely that the N-terminal extension may facilitate its interaction with an as yet undescribed protein to allow transfer into the PC. Since no endosymbiont-cytosoltargeted proteins have been characterized yet, nothing is presently known about the transfer of proteins through the second membrane into the PC in four-membraned plastids (McFadden 1999
; Wastl and Maier 2000
).
Origin of the Red Algalike Actin Gene: A New Source of Molecular Diversity
Taken together, our data provide strong evidence for the existence of a divergent, novel form of actin in P. helgolandii that originated from the nucleus (i.e., nucleomorph) of its red algal secondary symbiont. This gene was apparently transferred to the host genome, where it integrated, acquired regulatory sequences to drive its expression, and assumed some role in its new environment. While such a series of events may be complex, it should not be so surprising, because the host nucleus is known to contain a considerable number of genes encoding plastid-localized proteins, and these genes have all been transferred in much the same way (Gilson and McFadden 1997
; McFadden 1999
; Blanchard and Lynch 2000
). Moreover, it is well established that the eukaryotic hosts of prokaryotic endosymbiotic organelles (mitochondria and plastids) have also acquired genes from these endosymbiontsa specialized type of lateral transfer called "endosymbiotic gene replacement" (e.g., Keeling and Doolittle 1997
; Martin and Herrmann 1998
). The case of the red algal actin in P. helgolandii differs primarily in that a eukaryotic gene has been transferred to another, albeit distantly related, eukaryotic genome.
This actin lateral transfer has one other important characteristic of general significance. Unlike many endosymbiosis-associated gene transfers, in this case the red algalike actin did not simply replace the cryptophyte actin, but seems to have been integrated into the cryptophyte cytosol alongside the original protein. The relatively divergent sequence of the red algalike gene and its evolution of a novel 5'-terminus are consistent with this scenario. Our results show, therefore, that a host can reap the benefits of duplicate coding regions harbored in the symbiont genome in an entirely novel wayas a source of instant sequence diversity that has evolved under subtly, or even substantially, different selective pressures. Transferred genes with nonplastid functions have likely gone unnoticed until now because of the limited number of host nuclear genes that have been characterized from these alga. The red algalike actin in P. helgolandii may be the tip of an iceberg of endosymbiont-derived coding regions that turn up in the host genome of cryptophytes and other algae with secondary endosymbionts.
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Acknowledgements |
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Footnotes |
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1 Abbreviations: ABP, actin-binding protein; PC, periplastidial compartment.
2 Address for correspondence and reprints: Debashish Bhattacharya, Department of Biological Sciences, University of Iowa, 239 Biology Building, Iowa City, Iowa 52242.
3 Keywords: actin evolution
cryptophytes
symbiosis
gene transfer
nucleomorph
phylogeny
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literature cited |
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---|
Andersen, R. A., S. L. Morton, and J. P. Sexton. 1997. Provasoli-Guillard National Center for Culture of Marine Phytoplankton: 1997 list of strains. J. Phycol. 33(Suppl.):175.
Bhattacharya, D., and J. Ehlting. 1995. Actin coding regions: gene family evolution and use as a phylogenetic marker. Arch. Protistenkd. 145:155164.
Bhattacharya, D., and L. Medlin. 1995. The phylogeny of plastids: a review based on comparisons of small subunit ribosomal RNA coding regions. J. Phycol. 31:489498.[ISI]
. 1998. Algal phylogeny and the origin of land plants. Plant Physiol. 116:915.
Bhattacharya, D., S. K. Stickel, and M. L. Sogin. 1991. Molecular phylogenetic analysis of actin genic regions from Achlya bisexualis (Oomycota) and Costaria costata (Chromophyta). J. Mol. Evol. 33:525536.[ISI][Medline]
. 1993. Isolation and molecular phylogenetic analysis of actin coding regions from the prymnesiophyte alga, Emiliania huxleyi, using reverse transcriptase and PCR methods. Mol. Biol. Evol. 10:689703.[Abstract]
Bhattacharya, D., and K. Weber. 1997. The actin gene of the glaucocystophyte Cyanophora paradoxa: analysis of the coding region and introns and an actin phylogeny of eukaryotes. Curr. Genet. 31:439446.[ISI][Medline]
Blanchard, J. L., and M. Lynch. 2000. Organellar genes: why do they end up in the nucleus? Trends Genet.16:315320.
Bouget, F. Y., C. Kerbourc'h, M. F. Liaud, S. Loiseaux de Goer, R. S. Quatrano, R. Cerff, and B. Kloareg. 1995. Structural features and phylogeny of the actin gene of Chondrus crispus (Gigartinales, Rhodophyta). Curr. Genet. 28:164172.[ISI][Medline]
Branden, C., and J. Tooze. 1991. Introduction to protein structure. Garland, New York.
Delwiche, C. F., and J. D. Palmer. 1997. The origin of plastids and their spread via secondary symbiosis. Pp. 5386 in D. Bhattacharya, ed. Origins of algae and their plastids, Springer, Vienna.
Douglas, S. E., C. A. Murphy, D. F. Spencer, and M. W. Gray. 1991. Molecular evidence that cryptomonad algae are chimaeras of two phylogenetically distinct unicellular eukaryotes. Nature 350:148151.
Durnford, D. G., J. A. Deane, S. Tan, G. I. McFadden, E. Gantt, and B. R. Green. 1999. A phylogenetic assessment of the eukaryotic light-harvesting antenna proteins, with implications for plastid evolution. J. Mol. Evol. 48:5968.[ISI][Medline]
Felsenstein, J. 1978. Cases in which parsimony or compatibility methods will be positively misleading. Syst. Zool. 25:401410.
. 1997. PHYLIP manual. Version 3.572 P-PC. Department of Genetics, University of Washington, Seattle.
Fraunholz, M. J., E. Moerschel, and U.-G. Maier. 1998. The chloroplast division protein FtsZ is encoded by a nucleomorph gene in cryptomonads. Mol. Gen. Genet. 260:207211.[ISI][Medline]
Gilbert, D. 1992. SeqApp, A biosequence editor and analysis application, V1.9. Indiana University, Bloomington, Ind.
Gillott, M. A., and S. P. Gibbs. 1980. The cryptomonad nucleomorph: its ultrastructure and evolutionary significance. J. Phycol. 16:558568.[ISI]
Gilson, P. R., and G. I. McFadden. 1997. Good things in small packages: the tiny genomes of chlorarachniophyte endosymbionts. Bioessays 19:167173.
Greenwood, A. D. 1974. The Cryptophyta in relation to phylogeny and photosynthesis. Pp. 566567 in J. V. Sanders and D. J. Goodchild, eds. Electron microscopy 1974. Australian Academy of Science, Canberra, Australia.
Hendy, M. D., and D. Penny. 1989. A framework for the quantitative study of evolutionary trees. Syst. Zool. 38:297309.[ISI]
Herman, I. M. 1993. Actin isoforms. Curr. Opin. Cell Biol. 5:4855.[Medline]
Hibberd, D. J., and R. E. Norris. 1984. Cytology and ultrastructure of Chlorarachnion reptans (Chlorarachniophyta divisio nova, Chlorarachniophyceae classis nova). J. Phycol. 20:310330.[ISI]
Hofmann, C. J., S. A. Rensing, M. M. Hauber, W. F. Martin, S. B. Muller, J. Couch, G. I. McFadden, G. L. Igloi, and U.-G. Maier. 1994. The smallest known eukaryotic genomes encode a protein gene: towards an understanding of nucleomorph functions. Mol. Gen. Genet. 243:600604.[ISI][Medline]
Keeling, P. J., J. A. Deane, C. Hink-Schauer, S. E. Douglas, U.-G. Maier, and G. I. McFadden. 1999. The secondary endosymbiont of the cryptomonad Guillardia theta contains alpha-, beta-, and gamma-tubulin genes. Mol. Biol. Evol. 16:13081313.[Abstract]
Keeling, P. J., and W. F. Doolittle. 1997. Evidence that eukaryotic triosephosphate isomerase is of alpha-proteobacterial origin. Proc. Natl. Acad. Sci. USA 94:12701275.
Lopez, P. J., and B. Séraphin. 1999. Genomic-scale quantitative analysis of yeast pre-mRNA splicing: implications for splice-site recognition. RNA 5:11351137.
Ludwig, M., and S. Gibbs. 1989. Evidence that nucleomorphs of Chlorarachnion reptans (Chlorarachniophyceae) are vestigial nuclei: morphology, division and DNA-DAPI fluorescence. J. Phycol. 25:385394.[ISI]
McFadden, G. I. 1999. Plastids and protein targeting. J. Eukaryot. Mirobiol. 46:339346.
McFadden, G. I., P. R. Gilson, S. E. Douglas, T. Cavalier-Smith, C. J. Hofmann, and U.-G. Maier. 1997. Bonsai genomics: sequencing the smallest eukaryotic genomes. Trends Genet. 13:4649.[ISI][Medline]
McFadden, G. I., P. R. Gilson, and D. R. A. Hill. 1994. GoniomonasrRNA sequences indicate that this phagotrophic flagellate is a close relative of the host component of cryptomonads. Eur. J. Phycol. 29:2932.[ISI]
McKerracher, L., and S. P. Gibbs. 1982. Cell and nucleomorph division in the alga Cryptomonas. Can. J. Bot. 60:24402452.
Maddison, W. P., and D. R. Maddison. 1997. MacClade. Version 3.07. Sinauer, Sunderland, Mass.
Martin, W., and R. G. Herrmann. 1998. Gene transfer from organelles to the nucleus: how much, what happens, and why? Plant Physiol. 118:917.
Morrall, S., and A. D. Greenwood. 1982. Ultrastructure of nucleomorph division in species of Cryptophyceae and its evolutionary implications. J. Cell Sci. 54:311328.[ISI]
Oliveira, M., and D. Bhattacharya. 2000. Phylogeny of the Bangiophycidae (Rhodophyta) and the secondary endosymbiotic origin of algal plastids. Am. J. Bot. 87:482492.
Rymond, B. C., C. Pikielny, B. Seraphin, P. Legrain, and M. Rosbash. 1990. Measurement and analysis of yeast pre-mRNA sequence contribution to splicing efficiency. Methods Enzymol. 181:122147.[Medline]
Schlösser, U. G. 1994. SAGSammlung von Algenkulturen at the University of Göttingen. Bot. Acta 107:111186.
Sheterline, P., J. Clayton, and J. C. Sparrow. 1995. Actin. Protein Profile 2:2728.
Stiller, J. W., E. C. S. Duffield, and B. D. Hall. 1998. Amitochondriate amoebae and the evolution of DNA-dependent RNA polymerase II. Proc. Natl. Acad. Sci. USA 95:1176911774.
Stoltzfus, A., J. M. Logsdon Jr., J. D. Palmer, and W. F. Doolittle. 1997. Intron "sliding" and the diversity of intron positions. Proc. Natl. Acad. Sci. USA 94:1073910744.
Swofford, D. L. 1999. PAUP*: phylogenetic analysis using parsimony. Version 4.0b2. Smithsonian Institution, Washington, D.C.
Van de Peer, Y., S. A. Rensing, U.-G. Maier, and R. De Wachter. 1996. Substitution rate calibration of small subunit rRNA identifies chlorarachniophyte endosymbionts as remnants of green algae. Proc. Natl. Acad. Sci. USA 93:77327736.
Wastl, J., E. C. Duin, L. Iuzzolino, W. Dorner, T. Link, S. Hoffmann, H. Sticht, H. Dau, K. Lingelbach, and U.-G. Maier. 2000. Eukaryotically encoded and chloroplast located rubredoxin is associated with photosystem II. J. Biol. Chem. (in press).
Wastl, J., M. Fraunholz, S. Zauner, S. Douglas, and U.-G. Maier. 1999. Ancient gene duplication and differential gene flow in plastid lineages: the GroEL/Cpn60 example. J. Mol. Evol. 48:112117.[ISI][Medline]
Wastl, J., and U.-G. Maier. 2000. Transport of proteins into cryptomonad complex plastids. J. Biol. Chem. 275:2319423198.
Weber, K., and E. Kabsch. 1994. Intron positions in actin genes seem unrelated to the secondary structure of the protein. EMBO J. 13:12801286.[Abstract]
Zauner, S., M. Fraunholz, J. Wastl, S. Penny, M. Beaton, T. Cavalier-Smith, U. G. Maier, and S. Douglas. 2000. Chloroplast protein and centrosomal genes, a tRNA intron, and odd telomeres in an unusually compact eukaryotic genome, the cryptomonad nucleomorph. Proc. Natl. Acad. Sci. USA 97:200205.
Zhang, Z., B. R. Green, and T. Cavalier-Smith. 1999. Single gene circles in dinoflagellate chloroplast genomes. Nature 400:155159.