Department of Botany and Dean's Office, University of Guelph, Guelph, Ontario, Canada; and
Institute of Cellular and Molecular Biology, University of Texas at Austin
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Abstract |
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Introduction |
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Materials and Methods |
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RNA Extraction/cDNA Production in the Bangiales
RT-PCR was used to determine if introns were present in mature RNA or excised from all collections of Bangia and Porphyra listed in table 1
and the Bangia collections utilized in Müller et al. (1998)
. RNA was extracted following the procedure previously outlined for DNA extraction. Following this, 12 µl of extract was placed in a clean tube, to which 3 µl of 25 mM MgCl2 and 1 µl of DNAase was added. This mixture was then left at room temperature for 2 h, after which 5 µl was loaded in a 0.5% agarose gel to determine if all DNA had been digested within the extract. If this was the case, the extract was then immediately placed in the freezer at -20°C. RT-PCR using Titan by Boehringer Mannheim was used to amplify the RNA. The primers G02.1 and G09 (5'-ATC CAA GAA TTT CAC CTC TG-3') and G15.R (5'-GGA AGG AGG AGT CGT AAC AAG-3') and G07 were used to amplify the region containing the intron in positions 516 and 1506, respectively (Saunders and Kraft 1994
; Müller et al. 1998
). Following the protocols for RT-PCR outlined by Boehringer Mannheim, the reaction consisted of master mix 1 (1 µl of RNA template; 20 µM of each primer; 40 mM of dNTP; 2.5 µl DTT solution; dH2O) and master mix 2 (14 µl dH2O; 5 x RT-PCR buffer with MgCL2; 1 µl enzyme mix). These two master mixes were combined and placed in a 0.5-µl thin-walled PCR tube on ice. Amplification was performed as follows: initial denaturation at 94°C for 2 min; 10 cycles of denaturation at 94°C for 30 s, primer annealing at 50°C for 30 s, and extension for 2 min at 68°C; 25 cycles of denaturation at 94°C for 30 s, primer annealing at 50°C for 30 s, extension for 2 min at 68°C, plus cycle elongation for 5 s for each cycle; and a final extension of 7 min at 68°C. Sequencing of the amplified regions was carried out as previously outlined.
Alignment of Nuclear SSU rRNA Genes and Group IC1 Introns
The nuclear SSU rRNA gene and group IC1 intron sequences in the present study were incorporated into large alignments that spanned a large taxonomic range in order to ensure optimum alignment. The new sequences were manually aligned with the alignment editor AE2 (developed by T. Macke; see Larsen et al. 1993
) on the basis of sequence similarity and a previously established eukaryotic secondary-structure model (Gutell 1993
). The alignments were then subjected to a process of comparative sequence analysis (Gutell et al. 1985
). This process consisted of searching for compensating base changes using computer programs developed within the Gutell Laboratory (University of Texas at Austin, http://www.rna.icmb.utexas.edu/; discussed in Gutell et al. 1985
) and using the subsequent information to infer additional secondary-structural features. This refined alignment was reanalyzed and the entire process was repeated until the proposed structures were entirely compatible with the alignment. Secondary-structure diagrams were generated with the computer program XRNA (developed by B. Weiser and H. Noller, University of Santa Cruz). Individual secondary-structure diagrams will be available at http://www.rna.icmb.utexas.edu/, and alignments can be obtained from K.M.M.
Analysis of Nuclear SSU rRNA Gene and Group I Intron Sequences
All analyses on both the nuclear SSU rRNA gene and the group I introns were carried out using only well-aligned regions of the sequences. Parsimony analyses on the two introns were carried out with PAUP 3.1.1 (Swofford 1993
) with a heuristic search under the constraints of random sequence addition (100 replicates), steepest descent, and tree bisection-reconnection (TBR) branch swapping. The data were then subjected to bootstrap resampling (1,000 replicates). Analyses of the introns were carried out as follows: (1) the alignment gaps were treated as missing data, and (2) the alignment gaps were coded as independent single evolutionary events (insertion or deletion) based on secondary-structure models (Damberger and Gutell 1994). Group IC1 introns in Chlorella ellipsoidea (GenBank accession number A: X63520, B: D13324) were used as outgroups in the phylogenetic analyses. Parsimony trees could not be determined for the nuclear SSU rRNA gene due to the large number of taxa and limited computational capacity; however, neighbor-joining trees were calculated for this data set. PHYLIP (Felsenstein 1993
) was used to construct neighbor-joining trees for both the introns and the nuclear SSU rRNA genes using a matrix of distance values estimated according to the Kimura two-parameter model (Kimura 1980
) with a transition/transversion ratio of 2.0 and a single-category substitution rate, as well as the Jukes and Cantor (1969)
model (intron data set only). These data sets were also subjected to bootstrap resampling. Maximum-likelihood analysis was carried out using the puzzle function in PAUP (version 4.0 beta 2a) as described by Strimmer and von Haeseler (1996)
with 1,000 puzzling steps. The nuclear SSU rRNA gene sequence analyses of the Bangiales were run using Erythrotrichia carnea (L26189) and Erythrocladia sp. (L26188) as outgroups. These taxa were determined to be basal to the Bangiales and have been previously used as outgroup taxa for this order (Ragan et al. 1994
; Oliveira et al. 1995
; Müller et al. 1998
).
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Results |
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Structural Characteristics of Group IC1 Introns in the Bangiales
All of the rRNA introns in Bangia and Porphyra were determined to belong to the CI subgroup of the group I introns based on characteristic primary- and secondary-structure features (fig. 1 ) (Michel and Westhof 1990; Damberger and Gutell 1994). The lengths of these introns within the Bangiales varied considerably, ranging from 467 to 998 nt for intron 516 and ranging from 509 to 1,082 nt for intron 1506 (table 1
). Figure 1
is a consensus diagram of all of the group IC1 introns in the Bangiales. Both introns were observed to have large insertions within the P1 (12567 nt), P2 (62553 nt), P5 (391 nt), P6b (1084 nt), and P9.2 (553 nt) domains (fig. 1 ). The large insertions in the P1 and P2 domains were found in introns 1506 and 516, respectively (fig. 1
), whereas the other insertions (P5, P6b, and P9) were similar in size between both the 516 and the 1506 introns. The majority of the base pairs in the P4 and P7 helices are highly conserved (>95%) between both sets of introns, whereas the remaining helices are less conserved. The single positions and base pair "signatures" that distinguish the 516 and 1506 introns are shown as square boxes and dashed lines in figure 1
and detailed in table 3
. Approximately half of these nucleotides and the 5' positions of the base pairs noted in table 3
(and depicted in fig. 1
) are purines (A, G) in the 516 intron and are pyrimidines (C, U) in intron 1506 (positions 2, 25, 26, 97:277, 216:257, 217:256, 262:312, 322:326, and 335:364 [relative to Tetrahymena thermophila]). All of these positions are highly conserved (>95%) with the exception of base pair 97:277 (>90%, <95%). Position 172 and the 322:326 base pair have no homologous position with respect to T. thermophila. These three positions are highly conserved in both introns (>95%). Figure 2
highlights the differences between the 516 and 1506 introns in the P5b and P8 domains. The P5b helix (fig. 2a
) is one of the most distinct features of the 516 intron. This bifurcated helix, present in all of the Bangiales 516 introns, replaces the typical single helix present in all IC1 introns, including the Bangialean 1506 introns (fig. 2a
). The P8 domain (fig. 2b
) is also distinct, primarily on the basis of helix length. In the 516 version of this structure, the 3-bp helix is capped by a tetraloop, while the 1506 structure has a longer helix capped by a variable-length hairpin loop.
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Interestingly, some collections of B. fuscopurpurea that were identical with regard to nuclear SSU rRNA gene sequences were variable with regard to possession of either or both introns. For example, the collection from Greece contained both introns 516 and 1506; however, the other collection from Australia, with which it was identical, did not contain the 1506 intron. In addition, the collection from Ireland (IR) contained both introns 516 and 1506, whereas the other collections (WA and TX), with which it was nearly identical (differing by 8 bp) contained neither intron. A similar pattern occurred in B. fuscopurpurea from Massachussetts (MA), which did not contain the intron 1506, yet it was present in the other collections with nearly identical sequences (Helgoland and Nice, differing by 5 bp). Within Porphyra, this was only seen in one case: Porphyra yezoensis (HH) did not contain intron 1506, whereas P. yezoensis (OM) did.
Well-supported clades are along biogeographic lines with few exceptions, but the presence or absence of introns does not appear to be consistent based on geographic locations. For example, the well-supported clade (100% bootstrap) of B. fuscopurpurea containing primarily collections from the Atlantic and Mediterranean (NC, NJ, Greece, Helgoland, Nice, MA [with the exception of the Pacific samples from Australia and Mexico]) all contained intron 516, except the sample from Mexico, and the samples from Massachusetts and Australia did not contain intron 1506. Distromatic taxa (subgenus Diploderma) of Porphyra, P. miniata (CCAP 1379/2, NF) and P. amplissima (2), contained neither intron, whereas other species of Porphyra belonging to the subgenera Diplastida and Porphyra were variable in containing either intron.
Introns occur frequently within the Chlorophyta, but within the Rhodophyta, Bangia, Porphyra, and Hildenbrandia are the only genera currently known to contain introns (Ragan et al. 1993
). This trend raises the question of whether the introns within Hildenbrandia and the Bangiales might be related. Hence, figure 5
also includes the two 1506 introns from H. rubra and one chlorophyte algae, C. ellipsoidea (A, B). This figure presents the one most-parsimonious tree based on analysis of 1,025 parsimony-informative characters of well-aligned positions of the group IC1 intron in positions 516 and 1506. This tree has a length of 4,589 steps and a consistency index (CI) (a measure of the amount of homoplasy exhibited by the set of characters: for no homoplasy, CI = 1) of 0.4988. This data set was also analyzed by coding the gaps as single evolutionary events (e.g., 10 consecutive gaps are treated as one event rather than 10 separate events), whereas in the previous analysis, they were treated as missing data. This analysis depicted little change in the topologies of the trees; in fact, the resolution was much lower than when the gaps were treated as missing characters.
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Both the nuclear SSU rRNA gene NJ tree (fig. 4 ) and the phylogenetic tree using intron sequences (fig. 5 ) show similar trends. For example, the well-supported (100% bootstrap support; fig. 5 ) cluster containing B. fuscopurpurea from New Jersey (NJ), Nice, Australia, Greece, North Carolina (NC), Helgoland, and Massachusetts (MA) is also present in the intron phylogenetic tree for both the 1506 (100% MP bootstrap; 99% QPS; 92% NJ bootstrap) and 516 introns (98% MP bootstrap; <50% QPS; 70% NJ bootstrap) (fig. 5 ). In addition, the collections from Ireland, Newfoundland (NF), and B. fuscopurpurea (2) also cluster together within the nuclear SSU rRNA gene analysis (93% bootstrap) and the intron phylogeny for both intron insertion sites. This relationship is fairly well supported for the 1506 intron sequences (72% MP bootstrap; 94% QPS; 83% NJ bootstrap) but not for the 516 intron sequences (61% MP bootstrap; <50% QPS; <50% NJ bootstrap) (fig. 5 ).
Similarly, the 516 intron sequences within the freshwater taxon B. atropurpurea from the British Isles (BI12), Netherlands (NL), Great Lakes (GL), Italy (IT), and Ireland (IR) are positioned in a well-supported clade (100% MP bootstrap; 57% QPS; 85% NJ bootstrap), as are the sequences of the nuclear SSU rRNA gene for these collections. In addition, none of the freshwater collections contained intron 1506. The relationship of the marine collection from the Virgin Islands (VIS7) was unresolved (<55% bootstrap support) in both the intron analyses, and it is only weakly associated (62% bootstrap) with one of the main clades in the NJ nuclear SSU rRNA gene tree.
The relationship among the introns in Porphyra is not as well resolved as that in Bangia. However, some groupings are evident in all three analyses. For example, Porphyra sp. (SY) and P. pseudolinearis (TT) are closely associated in the nuclear SSU rRNA gene phylogeny (75% bootstrap), and the 516 intron sequences for these two taxa are also well associated (99% MP bootstrap; <50% QPS; 99% NJ bootstrap) (fig. 5
). In addition, the clade consisting of P. tenera (T1, TU-3), P. umbilicalis (NJ), P. yezoensis, and P. yezoensis (HH, OM) is present in the nuclear SSU rRNA gene phylogeny (although not well supported), and this cluster is also seen for the 516 introns for these taxa in figure 5 (100% MP bootstrap; 97% QPS; 76% NJ bootstrap). A close relationship among Porphyra rediviva, Porphyra sp. (Brest), and P. umbilicalis (HG) is reflected in both the nuclear SSU rRNA gene tree (100% bootstrap) and the 1506 portion of the intron phylogeny, with the relationship between P. rediviva and Porphyra sp. (Brest) being well supported (100% MP bootstrap; 100% QPS; 75% NJ bootstrap) and the 1506 intron sequence being only weakly associated with the previous clade (<50% MP bootstrap; 64% QPS; <50% NJ bootstrap). A grouping of Porphyra 1506 intron sequences consisting primarily of collections from Japan (P. yezoensis (OM, OG-1, OG-4, NA-2, NA-4), P. yezoensis narawaenis, P. tenera (KK, SK), P. pseudolinearis (TT), Porphyra sp. (SHH)) is moderately supported (82% MP bootstrap; 83% QPS; <50% NJ bootstrap). This trend is not, however, reflected in the nuclear SSU rRNA gene tree, where the relationship among many of the Japanese taxa is close but unresolved (<50% support). Many of the other relationships seen in the nuclear SSU rRNA gene NJ tree are similar to those in the intron trees, but most are not well supported by bootstrap analysis (<60%). The sequence divergence in the intron at position 516 varied considerably, ranging from 0% to as high as 31.0%. The sequence divergence for the intron in position 1506 was higher than that for the 516 intron (31.0% vs. 44.3%). Interestingly, the 1506 introns in P. spiralis var. amplifolia (Oliveira and Ragan 1994
) were quite distant from all other Porphyra and Bangia taxa, ranging from 30.3% to 44.3% sequence divergence.
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Discussion |
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The similarity in phylogenies between the 516 and 1506 introns and their nuclear SSU rRNA genes suggests that these introns have not been mobile during the evolution of the Bangiales. Introns have been documented in the nuclear-encoded rRNA genes of different Naegleria species. The majority of these occur at the 516 position of the nuclear SSU rRNA gene. A more extensive analysis of the Naegleria nuclear SSU rRNA genes revealed that this intron is absent in the majority of the species' nuclear SSU rRNA genes (De Jonckheere 1994
). De Jonckheere (1994)
hypothesized that since there was probably no selective advantage arising from the presence of these introns, they were eliminated in many of the descendants. In the case of the Bangiales, it appears that after initial acquisition of the introns in the ancestor of these taxa, there was subsequent vertical inheritance and evolution within the order (and species) and in some cases loss of the introns in either insertion site, as the intron phylogenies are very similar to that of the host gene. An alternative and less favorable hypothesis is that frequent lateral transfers within the order may have taken place, but this would result in intron phylogenies that would be widely discordant with the nuclear SSU rRNA gene phylogenies (Bhattacharya et al. 1994
).
The similarity among group I intron and nuclear SSU rRNA gene phylogenies has also been reported for other eukaryotic taxa. Takashima and Nakase (1997)
noted that introns had been transferred horizontally to distinct insertion sites in the yeast-like fungus Tilletiopsis flava and then inherited vertically. Similarly, introns 1506 and the nuclear SSU rRNA gene sequences share similar phylogenies in members of the green algal order Zygnematales (Bhattacharya et al. 1994
). Wilcox et al. (1992)
also postulated that the distribution of introns within other green algal taxa could be explained by inheritance of these sequences along with the gene. However, VanOppen, Olsen, and Stam (1993)
concluded that nuclear group I introns in green algae have arisen several times and do not appear to be lineage-specific and disagreed that the distribution of introns could be explained by vertical inheritance within the green algae as postulated by Wilcox et al. (1992)
. In the case of the Bangiales, the latter scenario appears to be possible. For instance, the intron phylogenies appear to be in accordance with the gene phylogenies, indicating long-term immobility and vertical inheritance.
The evolutionary history of the Bangiales is quite long, as bangiophyte taxa have been reported in the fossil record from the Proterozoic (1,200 MYA) (Butterfield, Knoll, and Swett 1990
), and the conchocelis stage of Porphyra has been reported in fossils up to 425 MYA (Campbell 1980
). In terms of utilizing the introns within the Bangiales as specific or geographic markers as speculated by Oliveira and Ragan (1994)
, the present study does not appear to indicate this possibility. In fact, analyses of the group I introns appear to provide further evidence for the synonymy of these two genera, as intron phylogenies are congruent with those of the nuclear SSU rRNA gene. This is not surprising, as other molecular studies using nuclear and chloroplast genes have indicated that these taxa are not monophyletic genera (Müller et al. 1998
). Thus, attempts at constructing a phylogenetic classification within the order based on morphology, life histories, and molecular analyses, and, in this case, intron sequences, have been problematic.
The observation that similar group I introns are distributed across different genomes and phylogenetically distant taxa is taken as evidence that intron transfer is relatively common (Burke 1988
). The clustering of group I introns in phylogenetic trees from distinct insertion sites suggests that these introns may be traced back to one or more events in which the introns were inserted into rRNA and vertically inherited or lost (Bhattacharya et al. 1994
; Bhattacharya 1998
). Within the Zygnematales, bootstrap analyses yielded little support for the single origin of all group I introns at different insertion sites within rRNA (Bhattacharya et al. 1994
). It is difficult to determine if this is the case with the introns within the Bangiales due to the lack of a suitable outgroup. Nonetheless, all of the 516 and 1506 introns form two separate clades distinct from each other. High sequence similarity between introns is consistent with descent from an ancestral intron that was initially acquired and vertically inherited (Schroeder-Diedrich, Fuerst, and Byers 1998
). In the protist Acanthamoeba, low sequence identity (highly variable) among introns in four different positions suggests that the intron acquisition occurred independently at the four positions after divergence of the taxa within the tree (Schroeder-Diedrich, Fuerst, and Byers 1998
). There is high sequence identity (0% sequence divergence in some cases) among many of the introns sequenced within the Bangiales; however, there is also considerable variability. For example, the 1506 introns in P. spiralis var. amplifolia (B, D, R) differ from all other intron sequences by 30%44% sequence divergence. Despite this, P. spiralis var. amplifolia is well supported as grouping with the remaining 1506 introns.
Homing-Endonuclease Pseudogenes in the Bangiales
The mobility of nuclear group I introns may depend on homing through the expression of intron-encoded homing endonucleases (Belfort and Roberts 1997
). The His-Cys box is one of several types of homing endonucleases that is restricted to group I introns (Johansen, Embley, and Willassen 1993
). Haugen et al. (1999)
described unusual P1 extensions within the nuclear SSU rRNA introns of Bangia and Porphyra. Their analysis showed that the intron sequences contained His-Cys box reading frames within the P1 extension and that these may represent homing-endonuclease pseudogenes. They also demonstrated that the ORF in the 1506 intron of P. spiralis var. amplifolia occurred on the strand complementary to that encoding the SSU rRNA gene and group I intron. The present study also observed this phenomenon for the P1 domain in the introns at position 1506 for P. fucicola and P. umbilicalis (HG), both of which also contained the His-Cys box motif noted in P. spiralis var. amplifolia. The introns in the 516 position that contained an ORF as well as a His-Cys box motif were within the P2 domain and not on the complementary strand, as seen in the 1506 introns. Haugen et al. (1999)
also noted frameshifts in the 3' end of the P. spiralis var. amplifolia intron ORF which resulted in a truncated C-terminal end and would explain the lack of cleavage activity in the in vitro translated protein. Haugen et al. (1999)
suggested that the truncation and frameshifts seen in the Bangialean intron ORFs may be the result of selection against endonuclease expression which would lethally cleave chromosomal DNA. Only two of the new sequences in this study had frameshifts, P. kanakaensis and B. fuscopurpurea (Helgoland), and all sequences were found to terminate prematurely. This may indicate that these ORFs are no longer functional; however, endonuclease activity has not been studied in these sequences and needs to be investigated.
Secondary-Structure Signatures of the Group IC1 Introns in the Bangiales
In 1987, Woese rationalized that the examination of higher-order structure from a phylogenetic perspective would enable us to examine the stages of evolution of rRNA structure and aid in extrapolating the significance of changes and variation in rRNA. Subsequently, Winker and Woese (1991)
delineated the three primary domains using structural characteristics of 16S rRNA. They defined two types of signature characters: (1) homologous positions whose compositions are (very nearly) constant (present in at least 94% of the representative sequences) within each of the domains or kingdoms and are characteristic of at least one, and (2) nonhomologous structures that characterize and distinguish the various groupings (e.g., structures that are present in one group and absent in another and/or structural units that differ strikingly from one group to another). Within these two types there are different examples of phylogenetically constrained elements ranging from single nucleotides and base pairs to hairpin loops, noncanonical pairing, insertions, deletions, and combinations of these elements, along with coaxial stacked helices or differences in numbers between homologous structures (Gutell 1992
).
In the present study, the Bangiales were observed to have sequence and structural elements, or "signatures," that differentiate these introns from each other as well as from other available intron sequences. As noted previously, the P5b domain in the 516 introns of the Bangiales contains a bifurcated helix that distinguishes these introns from all other group IC1 introns (fig. 2a ). This element is not present in the P5b domain in the 1506 bangialean introns and is an example of a nonhomologous signature. The variation in length in the P8 domain of the 1506 introns is also unique to the Bangiales and is not seen in the bangialean 516 introns. In addition to these structural elements, the introns in the Bangiales can be differentiated based on nonhomologous and homologous single-nucleotide and base pair signatures (table 3 ). For example, nucleotide 172 and base pair 322:326 have no homologous positions with respect to T. thermophila and can be considered nonhomologous signatures defining these introns. The two introns can be differentiated based on nucleotide and base pair signatures. For example, position 205 is a U in intron 516 (conserved in >95% of the sequences) and a C in intron 1506 (conserved in >95% of the sequences). These unique structural and sequence signatures provide further evidence that the introns in positions 516 and 1506 are probably the result of a single (although separate from each other) lateral transfer event and subsequent vertical inheritance. In addition, these structural signatures may provide a means for determining the possible origin of the Bangialean 516 and 1506 introns if an ancestral intron still exists. This information also yields a basis for utilizing introns to differentiate different phyla/organisms or to determine the origin of introns based on structural characteristics.
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
1 Keywords: Group IC1 introns
Bangiales
phylogeny
2 Address for correspondence and reprints: Robert G. Sheath, Dean's Office and Department of Botany, University of Guelph, Guelph, Ontario, Canada N1G 2W1. rsheath{at}uoguelph.ca
.
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References |
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