Symbiotic Hydra express a plant-like peroxidase gene during oogenesis
Zoological Institute, Christian-Albrechts-University Kiel, Olshausenstrasse 40, 24098 Kiel, Germany
* Author for correspondence (e-mail: tbusch{at}zoologie.uni-kiel.de)
Accepted 7 March 2005
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Summary |
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Key words: Hydra viridis, Chlorella sp., symbiosis, horizontal gene transfer, oogenesis, ascorbate peroxidase
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Introduction |
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Very little is known about the molecular basis that enables
Chlorella to survive and proliferate within the polyp's vacuoles and
controls the interaction between both partners. As described previously
(Habetha et al., 2003),
aposymbiotic Hydra viridis polyps deprived of their endosymbionts
grow normally when fed under laboratory conditions but show dramatically
reduced number of ovaries compared to symbiotic ones; testes formation appears
not to be affected by the absence of algae. Thus, symbiotic algae have severe
impact on sexual reproduction in Hydra viridis by promoting
oogenesis.
To understand the underlying genetic machinery we screened Hydra viridis for symbiosis-related genes using an unbiased approach based on cDNA representational difference analysis. Here, we show that one of these genes, HvAPX1, encodes an ascorbate peroxidase that is expressed exclusively during oogenesis. Sequence comparison shows that the gene is most closely related to plant peroxidases. Since the HvAPX1 gene, in contrast to orthologous genes in plants, does not contain introns, we discuss the possibility that during metazoan evolution it was translocated from a plant symbiont to the Hydra genome.
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Materials and methods |
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Nucleic acid preparation
For Hydra samples, total RNA was isolated by use of the Trizol
reagent (Invitrogen, Karlsruhe, Germany). After lysis of Hydra
tissue, symbiotic algae were removed by repeated centrifugation. As shown by
RT-PCR, algae could be removed quantitatively (see Fig. S1 in supplementary
material). The supernatants were subjected to the purification procedure
according to manufacturer's (Invitrogen) protocol. Chlorella RNA was
obtained with the RNeasy kit (Qiagen, Hilden, Germany) from isolated symbiotic
or pelleted free-living algae from liquid mass cultures. Genomic DNA from
Hydra was prepared using standard phenol/chloroform extraction and
from Chlorella with the DNeasy plant kit (Qiagen).
Subtraction of cDNA by representational difference analysis
Representational difference analysis (RDA) was used to identify genes with
different expression in symbiotic and aposymbiotic Hydra. We prepared
RNA samples from two sources: (i) synchronized mass cultures containing
asexual, testes-bearing and hermaphroditic H. viridis polyps without
algae (aposymbiotic) and (ii) with normal levels of algae. Double stranded
cDNA was generated using the Super Script System (Gibco-BRL, Gaithersburg,
USA). The three steps of RDA, (a) PCR generation of amplicons representative
of the starting populations of RNA molecules being compared, (b) the two-step
subtractive hybridization leading to the enrichment of amplified fragments of
differentially expressed genes and the sequential depletion of sequences
common to both populations; and (c) the purification, cloning and sequencing
of the resulting difference products were performed as described
(Hubank and Schatz, 1994).
Resulting DNA fragments were cloned, amplified by PCR and spotted on nylon
membranes for further analysis.
Isolation of ascorbate peroxidase genes from Chlorella
To be able to include sequence from the Chlorella symbionts in the
phylogenetic analysis, we cloned a cDNA fragment of Chlorella
ascorbate peroxidase from both symbiotic and free living Chlorella
(strain 211-11b) using PCR and primer set
5'-TTCGTCT(GC)G(GC)(AG)TGGCACG-3' and
5'-CCTGGAAGTAGG(AG)GTTGTC(AG)AA-3'. These primers cover conserved
regions of ascorbate peroxidase genes in various plant species including
Chlamydomonas sp?. The resulting PCR products were extended
by 5'- and 3'-RACE to fragments of 1080 bp (symbiotic
Chlorella) and 1055 bp (free living Chlorella). Comparison
of the two Chlorella ascorbate peroxidase-encoding fragments revealed
an identity of 98% at the cDNA level within 1000 bp of overlapping sequence.
Both fragments include the plant peroxidase domain and show high similarity to
known ascorbate peroxidases (e.g. expect value=5e-81 to ascorbate peroxidase
from wheat; Triticum aestivum).
Polymerase chain reaction amplifications
All PCRs were carried out with Taq polymerase (Amersham, Braunschweig,
Germany). For comparing the peroxidase domains between cDNA and genomic DNA of
Chlorella and Hydra the following primer were used:
5'-ATGCCGGCACGTACAGTGTTG-3' and
5'-CTTGAGCCACTCTACGGTCCAA-3' (for Chlorella);
5'-ATGATTGCTGGTACAGTTCGA-3' and
5'-CGTTATCAAGTATACTCGTTGG-3' (for Hydra). To amplify the
complete coding region of HvAPX1, we used the primer pair
5'-GTACAATGGTACCAAATAGAGT-3' and
5'-GAGGTAAATTAAAATATGTCTTCTG-3'.
Expression analysis by RT-PCR
Nucleic acid isolation and cDNA cloning were carried out as described
previously (Weinzinger et al.,
1994). The HvAPX1 fragment, corresponding to nucleotides 277-515
of the full-length cDNA of HvAPX1 was amplified with the primer set
5'-AAAACCAGGCAATGCTGGCTT-3',
5'-CTCTTGGTATATCAGAACTATCAA-3'. Hydra viridis actin was
amplified with 5'-CAATTTATGAAGGTTATGCTCTTC-3' and
5'-TATTTCCTTTCAGGTGGAGCAATA-3'.
Northern blot analysis
RNA was separated on a 1.3% agarose gel containing 2% formaldehyde and
transferred onto a nylon membrane (Hybond N). The hybridization probe of 239
bp was amplified with the same primers used for expression analysis of HvAPX1
by RT-PCR, gel purified and labelled with [-32P]dCTP.
Hybridization was performed in hybridization solution (50% formamide, 10 mmol
l-1 Tris-HCl, pH 7.5, 1% SDS, 1 x Denhardt's solution, 5
x SSC, 10% dextran sulphate) at 42°C overnight. Stringent washing
was performed: 2 x 10 min at room temperature in 2 x SSC/0.1% SDS
and 2 x 20 min in 0.2 x SSC/0.1% SDS at 65°C.
In situ hybridization
Whole-mount in situ hybridization was performed following standard
procedures (Endl et al., 1999)
using 239 bp digoxigenin-labelled RNA probes corresponding to nucleotides
277-515 of the full-length cDNA. For in situ hybridization of single
cell preparations, polyps were washed with 0.2 x PBS and 0.5 x
PBS. To obtain single cell suspensions, trypsinization was carried out with a
standard 10 x trypsin cell culture solution (Gibco-BRL) diluted to 0.5
x with 0.5 x PBS. After washing in chilled 1 x PBS suspended
cells were settled in a 1:1 mixture of 1 x PBS and 1 x HBSS
(Hanks' balanced salt solution; Gibco-BRL) and fixed on poly-L-lysine-coated
glass slides with formaldehyde. Slides with immobilized cells were subjected
to the standard in situ hybridization protocol as used for whole
mounts. Proteinase K treatment was for 2 min.
Peroxidase activity staining
Regions of peroxidase activity in polyps were stained as described
previously (Hoffmeister and Schaller,
1985) with the following modifications. Polyps were fixed in 4%
paraformaldehyde dissolved in 0.1 mmol l-1 sodium phosphate buffer,
pH 7.0. After fixation, the animals were washed in 0.1 mmol l-1
sodium phosphate buffer, pH 7.0, containing 5% sucrose. The animals were then
incubated for 15 min at room temperature in a solution containing an insoluble
substrate for the peroxidase, 20 mmol l-1
3,3'-diaminobenzidine (Sigma, Taufkirchen, Germany) and 0.003% (v/v)
hydrogen peroxide in PBS containing 0.1% Tween 20. Staining was stopped by
removing the solution and repeated washings in water and finally in ethanol
prior to embedding. L-ascorbate stock solution was made at 100 mmol
l-1 in 100 mmol l-1 sodium phosphate buffer, pH 7.0.
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Results |
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The completion of the Caenorhabditis elegans, Drosophila melanogaster, Ciona savignyi, mouse and human genome sequencing projects allows a comparison of peroxidase-related proteins from these organisms. By BLAST searches, the Hydra ascorbate peroxidase-related amino acid sequence could not be aligned with the translated genomes of these animals, nor with any animal amino acid sequence. Thus, the results strongly indicate that the predicted amino acid sequence of the Hydra viridis gene shares common sequence characteristics with plant ascorbate peroxidases and is distinct from animal peroxidases.
In order to determine the phylogenetic relationship of this unusual
Hydra viridis protein within the ascorbate peroxidase family, we
performed a genetic distance analysis. Fig.
2 illustrates the presumptive phylogenetic distances between the
Hydra putative ascorbate peroxidases and ascorbate peroxidases of
photosynthetic organisms from different phyla, including Trypanosoma,
which may have lost an ancient chloroplast
(Hannaert et al., 2003). The
analysis shows that the sequences encoded by the HvAPX1 group together with
those of plants and plant-like organisms. The phylogenetic comparison also
supports the view that the Hydra ascorbate peroxidase-encoding
sequence is distinct from the previously described putative peroxidases PPOD1
and PPOD2 from Hydra vulgaris
(Hoffmeister-Ullerich et al.,
2002
) and other heme-containing peroxidases, such as catalases,
found in animals.
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HvAPX1 expression is restricted to oogenesis
Initial RDA screening suggested that the HvAPX1 gene is upregulated in
symbiotic polyps compared to aposymbiotic polyps. This was confirmed by RT-PCR
using cDNAs from randomly selected aposymbiotic and symbiotic Hydra
viridis from our mass culture (Fig.
3A). To analyse whether gametogenesis affects expression of
HvAPX1, northern blot analysis was performed with RNA from three sources:
manually selected aposymbiotic and symbiotic polyps without any sign of sexual
differentiation, aposymbiotic and symbiotic polyps carrying testes, and
hermaphroditic aposymbiotic and symbiotic polyps having both testes and
ovaries (Fig. 3B). The analysis
with RNA from aposymbiotic and symbiotic Hydra viridis polyps
revealed the presence of a 1.3 kb HvAPX1 transcript only in animals undergoing
oogenesis. The northern analysis provided no evidence for the presence of
HvAPX1 transcripts in asexual animals and polyps undergoing spermatogenesis.
In polyps undergoing oogenesis, HvAPX1 transcripts can be detected in both
symbiotic and aposymbiotic polyps (Fig.
3B). The differential accumulation of HvAPX1 transcripts in
symbiotic polyps versus aposymbiotic polyps shown in
Fig. 3A most probably is due to
the fact that RNA for this RT-PCR experiment was isolated from two pools of
randomly picked polyps from the mass culture. In such cultures aposymbiotic
polyps develop ovaries only very rarely
(Habetha et al., 2003).
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The spatiotemporal expression pattern of HvAPX1 was visualized using
whole-mount and single cell in situ hybridization. As shown in
Fig. 4, HvAPX1 expression is
restricted to the ovary. The first HvAPX1-expressing cells are detectable at
an early stage of oogenesis, (Fig.
4A). At this stage the oocyte becomes determined and starts
phagocytosing the endocytes (Technau et
al., 2003). During later stages, when the oocyte incorporates
numerous endocytes, the number of HvAPX1-expressing cells rapidly increases
(Fig. 4B), and finally results
in a strongly stained oocyte (Fig.
4C). Transcript level rapidly decreases after the onset of
embryogenesis (data not shown). To determine in which cell types HvAPX1
transcripts are localized, we performed in situ hybridization on
suspended cells. As shown in Fig.
4D,E, HvAPX1 is expressed in interstitial cells/oogonia, as well
as in the oocyte itself.
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HvAPX1 in contrast to all known ascorbate peroxidases lacks introns
Plant genes encoding ascorbate peroxidases contain several introns, are
subjected to alternative mRNA splicing, and produce isoenzymes controlled by
tissue-specific or developmental signals
(Shigeoka et al., 2002). To
check for the presence of introns in both the Chlorella and Hydra
viridis ascorbate peroxidase genes, we performed RT-PCR and PCR on
genomic DNA using primers located at the beginning and end of the coding
regions, as well as primers flanking the peroxidase domain.
Fig. 5 shows that amplification
of the Chlorella peroxidase domain results in a smaller fragment from
cDNA and a larger one from genomic DNA. This indicates that in agreement with
other plant ascorbate peroxidase genes, the peroxidase domain in the
Chlorella gene is interrupted by intronic sequence of about 500 bp.
In contrast, when using Hydra cDNA and genomic DNA as template, PCR
amplifications resulted in an identical product of about 500 bp (lanes 3,4)
indicating that this region does not contain any intron. Moreover, when using
a primer pair flanking the complete coding region of HvAPX1, identical
fragments of about 1100 bp were obtained independently of cDNA or genomic DNA
being used as template (lanes 5,6). Thus, the Hydra HvAPX1 gene
appears to lack introns. The absence of introns was surprising, not only
because all known plant ascorbate peroxidase genes contain introns, but also
because all Hydra genes characterized so far at the genomic level
were found to contain introns (M.H. and T.C.G.B., personal observation). Thus,
HvAPX appears to have a rather unique gene structure.
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Discussion |
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How does a plant-related gene become incorporated into the Hydra nucleus?
Sequence comparison shows that HvAPX1 is closely related to plant
peroxidases and cannot be aligned with peroxidase-encoding genes from animals.
At least three different evolutionary scenarios appear possible.
First, HvAPX1 may have evolved from ancient precursors related either to
APX enzymes found in some protists or to cytochrome c peroxidases, which are
present in plants but also known from some fungi. The fact that such proteins
have not yet been reported from animals may be because of a deletion in the
animal lineage after divergence of the cnidarians. This idea would be
supported by finding of APX-related sequences in genomes of basal metazoans,
such as sponges, and of choanoflagellates which diverged before appearance of
the cnidarians. However, despite a relatively large body of databanks, there
is no evidence for the presence of APX-related genes in these taxa.
Interestingly, and supporting the view that ascorbate peroxidases are
restricted to plant-related organisms, phylogenetic analyses based on
mitochondrial sequences propose that APX-containing trypanosomes group
together with plants but not with animals
(Löytynoja and Milinkovitch,
2001).
Second, it has been proposed that the cnidarian-specific nematocytes
originated from free-living protists such as microsporidians whose genomes
were acquired early during jellyfish evolution
(Shostak and Kolluri, 1995;
Margulis and Sagan, 2002
). If
so, the origin of the Hydra APX gene may be the result of this
ancient fusion event.
Third, the HvAPX1 precursor was acquired early in evolution from an
organism not belonging to the animal lineage. The mechanisms by which genetic
material is transported within the cell are largely a matter of speculation;
possibilities that have been proposed for organelle gene transfer include bulk
DNA recombination and mRNA or cDNA (possibly virus-mediated) intermediates
(Henze and Martin, 2001;
Martin, 2003
). Given the
presence of several introns in all ascorbate peroxidase genes known in plants
(e.g. Shigeoka et al., 2002
),
the conspicuous absence of introns in HvAPX1 is at least consistent with the
view that this gene was translocated from an ancestral photosynthetic
endosymbiont to the Hydra genome via mRNA or cDNA
intermediates. Who was the donor? Since genome or EST data on basal organism
are rare, a detailed phylogenetic analysis of ascorbate peroxidases and
related proteins is not possible yet. However, the Chlorella enzyme
is less similar to HvAPX1 than those of other photosynthetic organisms such as
Euglena gracilis or Galdieria partita (Figs
1,
2). This makes it unlikely that
symbiotic Chlorella was the donor of this gene. Although the taxonomy
of the genus Hydra is complicated and still not settled, in a
phylogenetic analysis of the genus based on nuclear ribosomal DNA (D.
Martinez, personal communication) H. viridis appears as the basal
species. Performing TBLASTN searches of the Hydra
magnipapillata-expressed sequencing tag (EST) database
(www.hydrabase.org),
using the Hydra viridis HvAPX1 amino acid sequence as the query
sequence, resulted in identification of two non-overlapping Hydra
magnipapillata ESTs with high similarity to HvAPX1 (tac33e05.y1 with
expect value 7e-51; and taa92e12.x1 with expect value 8e-45) and one of which
contains the plant peroxidase domain (data not shown). Thus, the gene is
present and expressed not only in green but also in non-symbiotic species.
Moreover, since the EST library was prepared from asexual Hydra
magnipapillata polyps, APX, in species other than Hydra viridis
appears not to be restricted to oogenesis and may have taken on a different
function. A symbiotic relationship with algae is, under natural conditions,
observed only within Hydra viridis; transient symbiosis, however, can
experimentally be induced in non-symbiotic Hydra
(Rahat and Reich, 1984
;
Rahat, 1985
;
Rahat and Reich, 1986
)
indicating that the ability to form a symbiosis is a common feature of the
Hydra group. A symbiotic polyp, therefore, may best represent the
common ancestor.
A putative transfer of an APX gene may have happened early in
evolution from an ancient symbiont that got lost and was replaced in Hydra
viridis by Chlorella. This view is supported by (i) the fact
that Hydra viridis accepts other algae than Chlorella, at
least transiently, as symbionts (Pool,
1979; M.H., personal observation); and (ii) an increasing number
of reports indicating that loss of symbionts seems to have been common during
evolution (e.g. Hannaert et al.,
2003
; for a review, see
Cavalier-Smith, 2002
). Recent
evidence for loss of symbionts accompanied by subsequent colonization with
secondary symbionts as a response to dramatic changes in the ecosystem comes
from reef corals (Buddemeier and Fautin,
1993
; Baker, 2001
;
Baker et al., 2004
). Thus,
since the oldest known fossil cnidarians were found in marine sediments, the
early evolution of the Hydra group from marine to freshwater
habitants may have been accompanied by an exchange of endosymbionts.
What is the possible function of HvAPX1 in Hydra viridis oogenesis?
Peroxidase activity is known to coincide with oogenesis and embryogenesis
in Hydra (Technau et al.,
2003). During egg formation and embryo development, cells of the
interstitial cell lineage aggregate and proliferate so that the ovary is
visible as a swelling on the upper body column. These interstitial cells
become highly motile and invade the cytoplasm of the oocyte. It can be assumed
that this process requires signalling, which induces and controls the cell
movements and incorporation into the egg, as well as local interaction with
the surface of the oocyte during ingestion. There is evidence that reactive
oxygen species (ROS) are involved in cell signalling, proliferation and
pattern formation in Cnidaria (Blackstone,
2003
). Thus, HvAPX1 may take part in the regulation of the
processes during ovary formation and oogenesis by balancing the redox state at
an optimal level. Alternatively, since high doses of reactive oxygen species
(ROS), as well as lack of antioxidants, have severe negative impact on gametes
in human and rats (e.g. Aziz et al.,
2004
), HvAPX1 may act as a ROS-scavenger, protecting the
developing egg from oxidative damage. Similarly, Technau and co-workers
suggest that peroxidase activity during oogenesis and embryogenesis may
protect the incorporated cells in the oocyte from rapid apoptotic degradation
(Technau et al., 2003
).
Transfer of genes from endosymbionts to hosts is well known from
mitochondrial and plastidal genes, resulting in a centralization of the
genetic material within the nucleus. Moreover, recent data show that genes
originally belonging to the symbiont can be subject to secondary transfer
events between different species (e.g.
Bergthorsson et al., 2003;
Won and Renner, 2003
). It is
intriguing to speculate that transferred genes are involved in oocyte
formation to ensure the survival of the symbiont. During later stages of
symbiogenesis the function of this gene may even be transferred to a secondary
symbiont.
Taken together, the work described here represents the first step in determining the molecular changes associated with symbiosis in Hydra. The observations may have far-reaching evolutionary implications and provide evidence that the evolution of genomes in basal metazoa may be much more dynamic than previously thought.
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Acknowledgments |
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Footnotes |
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References |
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---|
Aziz, N., Saleh, R. A., Sharma, R. K., Lewis-Jones, I., Esfandiari, N., Thomas, A. J., Jr and Agarwal, A. (2004). Novel association between sperm reactive oxygen species production, sperm morphological defects, and the sperm deformity index. Fertil. Steril. 81,349 -354.[CrossRef][Medline]
Baker, A. C. (2001). Reef corals bleach to survive change. Nature 411,765 -766.[CrossRef][Medline]
Baker, A. C., Starger, C. J., McClanahan, T. R. and Glynn, P. W. (2004). Coral reefs: corals' adaptive response to climate change. Nature 43,741 .
Bergthorsson, U., Adams, K. L., Thomason, B. and Palmer, J. D. (2003). Widespread horizontal transfer of mitochondrial genes in flowering plants. Nature 424,197 -201.[CrossRef][Medline]
Blackstone, N. W. (2003). Redox signaling in
the growth and development of colonial hydroids. J. Exp.
Biol. 206,651
-658.
Buddemeier, R. W. and Fautin, D. G. (1993). Coral bleaching as an adaptive mechanism. Bioscience 43,320 -326.
Bushman, F. (2002). Lateral DNA Transfer: Mechanisms and Consequences. Cold Spring Harbor Laboratory Press. Cold Spring Harbor, New York.
Campbell, R. D. (1990). Transmission of symbiotic algae through sexual reproduction in Hydra: movement of algae into the oocyte. Tissue Cell 22,137 -147.[CrossRef]
Cavalier-Smith, T. (2002). Chloroplast evolution: secondary symbiogenesis and multiple losses. Curr. Biol. 12,R62 -R64.[CrossRef][Medline]
Endl, I., Lohmann, J. U. and Bosch, T. C. G.
(1999). Head-specific gene expression in Hydra:
complexity of DNA-protein interactions at the promotor of ks1 is inversely
correlated to the head activation potential. Proc. Natl. Acad. Sci.
USA 96,1445
-1450.
Friedl, T. (1997). The evolution of green algae. In The Origins of Algae and their Plastids (ed. D. Bhattacharya), pp. 87-101. Berlin: Springer-Verlag.
Habetha, M., Anton-Erxleben, F., Neumann, K. and Bosch, T. C. G. (2003). The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts. Zoology 106,101 -108.
Hannaert, V., Saavedra, E., Duffieux, F., Szikora, J. P.,
Rigden, D. J., Michels, P. A. and Opperdoes, F. R. (2003).
Plant-like traits associated with metabolism of Trypanosoma parasites.
Proc. Natl. Acad. Sci. USA
100,1067
-1071.
Henze, K. and Martin, W. (2001). How do mitochondrial genes get into the nucleus? Trends Genet. 17,383 -387.[CrossRef][Medline]
Hoffmeister, S. A. and Schaller, H. C. (1985). A new biochemical marker for foot-specific cell differentiantion in Hydra. Roux's Arch. Dev. Biol. 194,453 -461.[CrossRef]
Hoffmeister-Ullerich, S. A., Herrmann, D., Kielholz, J.,
Schweizer, M. and Schaller, H. C. (2002). Isolation of a
putative peroxidase, a target for factors controlling foot-formation in the
coelenterate Hydra Eur. J. Biochem.
269,4597
-4606.
Hubank, M. and Schatz, D. G. (1994). Identifying differences in mRNA expression by representational difference analysis of cDNA. Nucleic Acids Res. 22,5640 -5648.[Abstract]
Huss, V. A. R., Holweg, C., Seidel, B., Reich, V., Rahat, M. and Kessler, E. (1993/94). There is an ecological basis for host/symbiont specificity in Chlorella/Hydra symbioses. Endocytobiosis Cell Res. 10, 35-46.
Itoh, T., Martin, W. and Nei, M. (2002).
Acceleration of genomic evolution caused by enhanced mutation rate in
endocellular symbionts. Proc. Natl. Acad. Sci. USA
99,12944
-12948.
Lenhoff, H. M. and Muscatine, L. (1963). On the role of algae symbiotic with Hydra. Science 142,956 -958.
Löytynoja, A., and Milinkovitch, M. C.
(2001). Molecular phylogenetic analyses of the mitochondrial
ADP-ATP carriers: the Plantae/Fungi/Metazoa trichotomy revisited.
Proc. Natl. Acad. Sci. USA
98,10202
-10207.
Margulis, L. and Sagan, D. (2002). Acquiring Genomes: A Theory of the Origin of Species. New York: Basic Books.
Martin, W. (2003). Gene transfer from
organelles to the nucleus: frequent and in big chunks. Proc. Natl.
Acad. Sci. USA 100,8612
-8614.
Martin, W., Rujan, T., Richly, E., Hansen, A., Cornelsen, S.,
Lins, T., Leister, D., Stoebe, B., Hasegawa, M. and Penny, D.
(2002). Evolutionary analysis of Arabidopsis,
cyanobacterial, and chloroplast genomes reveals plastid phylogeny and
thousands of cyanobacterial genes in the nucleus. Proc. Natl. Acad.
Sci. USA 99,12246
-12251.
McAuley, P. J. (1985). Regulation of numbers of symbiotic Chlorella in digestive cells of green Hydra. Endocyt. Cell Res. 2,179 -190.
Miller, M. A., Technau, U., Smith, K. M. and Steele, R. E. (2000). Oocyte development in Hydra involves selection from competent precursor cells. Dev. Biol. 224,326 -338.[CrossRef][Medline]
Mochizuki, K., Sano, H., Kobayashi, S., Nishimiya-Fujisawa, C. and Fujisawa, T. (2000). Expression and evolutionary conservation of nanos-related genes in Hydra. Dev. Genes Evol. 210,591 -602.[CrossRef][Medline]
Muscatine, L. (1983). Isolating endosymbiotic algae from Hydra viridis. In Hydra: Research Methods (ed. H. M. Lenhoff), pp.391 -392. New York: Plenum Press.
O'Brien, T. L. (1982). Inhibition of vacuolar membrane fusion by intracellular symbiotic algae in Hydra viridis (Florida strain). J. Exp. Zool. 223,211 -218.[Medline]
Ochman, H. and Moran, N. A. (2001). Genes lost
and genes found: evolution of bacterial pathogenesis and symbiosis.
Science 292,1096
-1098.
Palenik, B. (2002). The genomics of symbiosis:
hosts keep the baby and the bath water. Proc. Natl. Acad. Sci.
USA 99,11996
-11997.
Pardy, R. L. (1983). Preparing aposymbiotic hydra. In Hydra: Research Methods (ed. H. M. Lenhoff), pp 394-395. New York: Plenum Press.
Pool, R. R. (1979). The role of algal antigenic determinats in the recognition of potential algal symbionts by cells of chlorohydra. J. Cell Sci. 35,367 -379.[Abstract]
Rahat, M. (1985). Competition between chlorellae in chimeric infections of Hydra viridis: the evolution of a stable symbiosis. J. Cell Sci. 77, 87-92.[Abstract]
Rahat, M. and Reich, V. (1984). Intracellular infection of aposymbiotic Hydra viridis by a foreign free-living Chlorella sp.: initiation of a stable symbiosis. J. Cell Sci. 65,265 -277.[Abstract]
Rahat, M. and Reich, V. (1986). Algal endosymbionts in brown hydra: host/symbiont specificity. J. Cell Sci. 86,273 -286.[Abstract]
Saitou, N. and Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,406 -425.[Abstract]
Shigeoka, S., Ishikawa, T., Tamoi, M., Miyagawa, Y., Takeda, T.,
Yabuta, Y. and Yoshimura, K. (2002). Regulation and function
of ascorbate peroxidase isoenzymes. J. Exp. Bot.
53,1305
-1319.
Shostak, S., and Kolluri, V. (1995). Symbiotic origins of cnidarian cnidocysts. Symbiosis 19, 1-19.
Technau, U., Miller, M. A., Bridge, D. and Steele, R. E. (2003). Arrested apoptosis of nurse cells during Hydra oogenesis and embryogenesis. Dev. Biol. 260,191 -206.[CrossRef][Medline]
Thorington, G. and Margulis, L. (1981). Hydra viridis: transfer of metabolites between Hydra and symbiotic algae. Biol. Bull. 160,175 -188.[Abstract]
Thorington, G., Berger, B. and Margulis, L. (1979). Transmission of symbionts through the sexual cycle of Hydra viridis. I. Observations on living organisms. Trans. Amer. Microsc. Soc. 98,401 -413.
Timmis, J. N., Ayliffe, M. A., Huang, C. Y. and Martin, W. (2004). Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat. Rev. Genet. 5, 123-135.[CrossRef][Medline]
Weinzinger, R., Salgado, L. M., David, C. N. and Bosch, T. C.
G. (1994). Ks1, an epithelial cell-specific gene, responds to
early signals of head formation in Hydra.
Development 120,2511
-2517.
Wilkinson, S. R., Obado, S. O., Mauricio, I. L. and Kelly, J.
M. (2002). Trypanosoma cruzi expresses a plant-like
ascorbate-dependent hemoperoxidase localized to the endoplasmic reticulum.
Proc. Natl. Acad. Sci. USA
99,13453
-13458.
Won, H. and Renner, S. S. (2003). Horizontal
gene transfer from flowering plants to Gnetum. Proc. Natl. Acad.
Sci. USA 100,10824
-10829.
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