Operon Structure and Trans-Splicing in the Nematode Pristionchus pacificus

Kwang-Zin Lee and Ralf J. Sommer

Max-Planck Institut für Entwicklungsbiologie, Abteilung für Evolutionsbiologie, Tübingen, Germany

Correspondence: E-mail: ralf.sommer{at}tuebingen.mpg.de.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
In the nematode Caenorhabditis elegans, up to 15% of the genes are organized in operons. Polycistronic precursor RNAs are processed by trans-splicing at the 5' ends of genes by adding a specific trans-spliced leader. Ten different spliced leaders are known in C. elegans that differ in sequence and abundance. The SL1 leader is most abundant and is spliced to the 5' ends of monocistronic genes and to upstream genes in operons. Trans-splicing is common among nematodes and was observed in the genera Panagrellus, Ascaris, Haemonchus, Anisakis, and Brugia. However, little is known about operons in nonrhabditid nematodes. Dolichorhabditis CEW1, another rhabditid nematode that is now called Oscheius CEW1, contains operons and SL2 trans-splicing. We have studied the presence of operons and trans-splicing in Pristionchus pacificus, a species of the Diplogastridae that has recently been developed as a satellite organism in evolutionary developmental biology. We provide evidence that P. pacificus contains operons and that downstream genes are trans-spliced to SL2. Surprisingly, the one operon analyzed so far in P. pacificus is not conserved in C. elegans, suggesting unexpected genomic plasticity.

Key Words: Pristionchus pacificusCaenorhabditis elegans • operons • trans-splicing • aldose reductase


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
The free-living nematode Caenorhabditis elegans is unusual among eukaryotes in that many genes are organized in operons, a form of gene organization commonly seen in eubacteria and archaea. It has been suggested that the C. elegans genome contains around 1,000 operons with around 15% of the genes being organized in this way (Blumenthal et al. 2002). Polycistronic precursor RNAs contain between two and eight genes each and are processed by trans-splicing at the 5' ends of genes, followed by polyadenylation at the 3' ends of genes. Intercistronic spaces are usually between 100 to 400 nt (Blumenthal et al. 2002). There are up to 10 different trans-spliced leaders that are spliced to the 5' ends of genes (Blumenthal et al. 2002). Although all spliced leaders are 22 nt in length, they differ in sequence and abundance. The spliced leader SL1 is most abundant and is spliced to the 5' ends of monocistronic genes and to the upstream genes in operons. Downstream genes in operons rarely contain an SL1 leader but usually contain one of several other leader sequences, such as SL2 (Blumenthal 1995). In contrast to SL1, SL2 and other spliced leaders are exclusively trans-spliced to downstream genes in operons (Spieth et al. 1993). Taken together, C. elegans exhibits two features, trans-splicing and polycistronic gene organization in operons that are not commonly seen in multicellular animals.

Previous studies had shown that one of these features, trans-splicing, is common among nematodes and not restricted to C. elegans. In particular, Caenorhabditis briggsae, Panagrellus redivivus, Ascaris suum, Haemonchus contortus, Anisakis spp. (Bektesh, Van Doren, and Hirsh 1988) and Brugia malayi (Takacs et al. 1988) contain SL1 splicing. The observation of SL1 splicing in such a wide variety of nematodes strongly suggests that trans-splicing is an ancient character of nematodes. In contrast, little is known about operons in nonrhabditid nematodes. For nematodes outside of the genus Caenorhabditis, Evans et al. (1997) have shown that Dolichorhabditis CEW1 (this species is now called Oscheius sp.1 CEW1 and will in the following be called Oscheius/Dolichorhabditis sp.1 CEW1) contains operons and SL2 trans-splicing. Oscheius/Dolichorhabditis sp.1 CEW1 is another rhabditid nematode and is closely related to C. elegans when compared with the other species mentioned above (fig. 1). Thus, the current picture suggests that trans-splicing and the SL1 leader sequence are conserved among the major branches of nematodes, whereas information on SL2 splicing and gene organization in operons is limited to the two genera Caenorhabditis and Oscheius of the Rhabditidae. To further evaluate the occurrence of trans-splicing and operon organization we studied another free-living species, Pristionchus pacificus (fig. 1).



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FIG. 1. Phylogenetic relationship of five representative nematode species that have been compared for the existence of operons and trans-splicing. Caenorhabditis elegans and Oscheius/Dolichorhabditis sp.1 CEW1 are members of the Rhabditidae. Both species contain operons, whereas clustering of the SL1 RNA genes has just been observed in C. elegans. Pristionchus pacificus belongs to the Diplogastridae and is thought to have separated from C. elegans for approximately 100 to 200 Myr. Panagrellus redivivus and Ascaris suum belong to the Panagrolaimidae and Ascarididae, respectively. SL1 RNA has been observed in both species using a C. elegans SL1 hybridization probe. It is unknown if these species contain operons and SL2 splicing

 
P. pacificus belongs to the Diplogastridae family and has recently been developed as a satellite organism for functional studies in evolutionary developmental biology (for review see Sommer [2000]). The comparison of P. pacificus to the model organism Caenorhabditis elegans allows evolutionary alterations of developmental processes to be identified that result in cellular and morphological changes. One developmental process that has been studied in great detail is the development of the vulva, the egg-laying structure of nematode females and hermaphrodites (Sommer et al. 1996; Eizinger, Jungblut, and Sommer 1999). Genetic and molecular studies in P. pacificus have recently been complemented by a genomic approach (Srinivasan et al. 2002). A BAC library was generated and completely end sequenced. A total of 133 polymorphisms were generated from BAC end and expressed sequence tag (EST) sequences (Srinivasan et al. 2002). These markers were tested on a meiotic mapping panel, providing a first genetic linkage map of P. pacificus, which was complemented by the generation of a physical map (Srinivasan et al. 2003).

The molecular analysis of P. pacificus vulval patterning genes such as mab-5, the Antennapedia homolog of nematodes, had already indicated the presence of SL1 splicing of monocistronic genes (Jungblut and Sommer 1998). Here, we show that P. pacificus contains operons and that downstream genes are trans-spliced to spliced leaders other than SL1. The genes found in this operon are however, not conserved between P. pacificus and C. elegans. Operons and trans-splicing to SL2-like leaders are shared features among the nematode families Rhabditidae and Diplogastridae, the last common ancestor of which lived approximately 200 MYA.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Standard molecular biological procedures, such as Southern blots, were performed as described (Sambrook and Russell 2001).

Ppa-ar-1 Cloning
The aldose reductase from P. pacificus was cloned by nested PCR using the degenerate primers 5'-ATGCCIYTNATHGGIATNGGNACITGGAC-3' in the N-terminal and 5'-RAARTGIGGRTGRTAYTCIACYTG-3' in the C-terminal region in the first-round PCR and 5'-GGNTAYMGITTYATMGAYACIGCICA-3' in the N-terminal and 5'-DATYTCRAARTTNSWIACICC-3' in the C-terminal region in the second-round PCR. To clone the 5' and 3' regions of Ppa-ar-1, including the SL2 leader sequence, we used the GeneRacer Kit (Invitrogen) following the manufacturer's instructions. The GenBank accession number for the Ppa-ar-1 sequence and the other P. pacificus sequences reported in this study are AY286498 to AY286500.

Cloning Pristionchus SL1 and SL2 Genes
We used the genome walking strategy (Siebert et al. 1995) and the Genome Walker Kit (Clontech) to clone SL2-like genes from P. pacificus. The obtained genomic fragments were sequenced and used for screening the P. pacificus HindIII BAC library. BAC clones were placed on the physical map and the genetic linkage map by generating a single-strand conformation polymorphism (SSCP) between the wild-type strain P. pacificus var. California and the polymorphic strain P. pacificus var. Washington as described previously (Srinivasan et al. 2002). In the case of SL1, a labeled oligonucleotide was used directly as a probe for screening the P. pacificus HindIII BAC library.

BAC Clone Preparation and Sequencing
PPBAC-21E22 was sequenced using the shotgun approach (Fleischmann et al. 1995). BAC DNA was sheared, blunt-end cloned into the pCR4-TOPO vector and transformed into E. coli (Shotgun Subcloning Kit, Invitrogen). The plasmid DNA from the shotgun library was prepared using Quiaprep Miniprep (Qiagen). Sequencing reactions were performed by using the BigDye sequencing Kit (version 2) (AppliedBiosystems) on an ABI prism 3700.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Cloning of the P. pacificus Aldose Reductase Gene
Two of the three major cell death regulator genes in C. elegans are organized in operons. The bcl-2 homolog ced-9 is organized in an operon with the cytochrome cyt-1 (Hengartner and Horvitz 1994), whereas the adaptor gene ced-4 is part of the complex operon CEOP3224 with three novel proteins and the aldose reductase gene C35D10.6 (fig. 2A) (Blumenthal et al. 2002). Among other candidates, we investigated whether the P. pacificus orthologs of these cell death regulators are also organized in operons. Ppa-cyt-1 was obtained in a large-scale EST project (McCarter et al. 2002). Genomic sequencing of the Ppa-cyt-1 region revealed that this gene is not part of an operon and that no gene with sequence similarity to Cel-ced-9 is physically linked to Ppa-cyt-1 (data not shown).



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FIG. 2. Operon structure and gene organization in P. pacificus and C. elegans. (A) C. elegans linkage group III and the location and organization of the C35D10.6 operon. C35D10.6 is the last gene in an operon containing the cell death adaptor molecule ced-4 (C35D10.9). (B) Organization of the identified P. pacificus operon containing the C35D10.6-like aldose reductase Ppa-ar-1. The three C. elegans orthologs of the genes found in the P. pacificus operon are spread over a large part of C. elegans chromosome III. (C, D, E) Sequence alignment of Ppa-ar-1 and C35D10.6 (C), the Ppa-ZK1128.4-like gene and ZK1128.4 (D), and the Ppa-R144.2-like gene and R144.2 (E), respectively. Identical amino acids are shown in black. (F) Trans-splicing specificity of the genes in the P. pacificus operon. RT-PCR reactions have been performed using 3' primers specific for R144.2b (lanes 1 to 4), ZK1128.4 (lanes 5 to 8), and C35D10.6 (lanes 9 to 12) and 5' primers representing the different spliced leaders SL1 (lanes 2, 6, and 10), SL2a (lanes 3, 7, and 11), and SL2b (lanes 4, 8, and 12). Lane 1,5, and 9 represent negative controls. Ppa-R144.2b is only trans-spliced to SL1. In contrast, both downstream genes are trans-spliced to SL1 and SL2-like leaders, a phenomenon also known from C. elegans.

 
In an effort to clone the cell death regulator Ppa-ced-4 by an "operon-synteny approach," we performed RT-PCR experiments to amplify the downstream aldose reductase gene C35D10.6. A fragment of an aldose reductase gene in P. pacificus was obtained that has high sequence similarity to C35D10.6 (fig. 2C). When compared with the complete set of aldose reductase genes from C. elegans, the fragment of this P. pacificus gene has the highest sequence similarity to C35D10.6. Nonetheless, this observation does not rule out other aldose reductase genes in P. pacificus that are more similar to C35D10.6. Therefore, we designated the obtained gene as Ppa-ar-1. C35D10.6 and Ppa-ar-1 have an overall sequence identity of 46% at the amino acid level (fig. 2C). The Ppa-ar-1 cDNA fragment was used to probe the P. pacificus BAC library to obtain a full-length clone and to study the genomic localization and organization. Ppa-ar-1 was identified on several BAC clones, one of which, PPBAC-14C19, was chosen for further characterization.

Next, we generated an SSCP polymorphism in PPBAC-14C19 to obtain the position of the Ppa-ar-1 gene in the P. pacificus genome by placing it on the meiotic mapping panel (Srinivasan et al. 2002). S197 was generated in the PPBAC-14C19z end and was shown to be located on chromosome III. S197 has the same linkage map position as S1, an SSCP polymorphism generated in the Ppa-pal-1 gene indicating that Ppa-ar-1 and Ppa-pal-1 are linked to one another. This observation is similar to C. elegans, where C35D10.6 and pal-1 are also linked. In addition to the high sequence similarity between Ppa-ar-1 and C35D10.6, the comparable genomic position provides further evidence that Ppa-ar-1 is the 1:1 ortholog of the C. elegans C35D10.6 gene.

Ppa-ar-1 Is a Downstream Gene in an Operon and Is Trans-Spliced to an SL2-like Leader
Most of the genes in C. elegans operons are separated from one another by approximately 100 bp (Blumenthal et al. 2002). To determine if Ppa-ar-1 is also part of an operon, we sequenced the neighboring region on PPBAC-14C19. Approximately 100 bp upstream of Ppa-ar-1, we identified another open reading frame (ORF) that is most similar to the C. elegans gene ZK1128.4 (fig. 2B and D). ZK1128.4 encodes a presumptive transcription factor with high sequence similarity to tfb4 (information available from WormBase: http://www.wormbase.org/). ZK1128.4 in C. elegans is also located on chromosome III but is not part of the same operon as C35D10.6 (fig. 2A).

Next we wanted to provide further evidence that Ppa-ar-1 and the Ppa-ZK1124.8-like gene are part of an operon. If so, the downstream genes in the operon should most likely be trans-spliced to a spliced leader other than SL1. While the sequence of the SL1 leader is conserved among nematodes, the comparison between C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1 indicated that SL2-like sequences differ between species. Therefore, RT-PCR experiments with Ppa-ar-1 specific primers on oligo-capped full-length mRNA of P. pacificus were performed (Maruyama and Sugano 1994). This strategy provides a simple way of identifying the sequence of the spliced leader. The determined sequence was identical to the genomic sequence of Ppa-ar-1 and was preceded by the sequence 5'-GGATTTCATCCCATATCTCAAG-3', which is related, but not exactly identical, to the SL2 sequence of C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1. We designated this spliced leader as Ppa-SL2a and confirmed that Ppa-ar-1 is SL2a spliced by using an SL2a-specific primer in a RT-PCR experiment (fig. 2F). These results suggest that P. pacificus has operons and that, like in C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1, downstream genes are trans-spliced to an SL2-like leader.

The Genes in the Ppa-ar-1 Operon Are Not Homologous to the Genes in the CEOP3224 Operon of C. elegans
To determine if the Ppa-ZK1128.4-like gene is the most upstream gene of this P. pacificus operon and therefore SL1 spliced, RT-PCR experiments with oligo-capped full-length mRNA for the Ppa-ZK1128.4-like gene were carried out. Surprisingly, Ppa-ZK1128.4 is trans-spliced to another SL2-like leader sequence that we have designated Ppa-SL2b (fig. 2F). This result indicates that, first, P. pacificus contains several additional spliced leaders, such as C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1 and, second, that the Ppa-ZK1128.4-like gene is not the most upstream gene of this operon. Genomic sequencing on PPBAC-14C19 revealed the presence of another gene that is located approximately 400 bp upstream of the Ppa-ZK1128.4-like gene (fig. 2B). This gene has sequence similarity to the C. elegans gene R144.2b, which is located on chromosome III but in a region other than C35D10.6 and ZK1128.4 (fig. 2A and E). C. elegans R144.2b contains sequence similarity to a transcriptional repressor in yeast with proline-rich zinc fingers (information available from WormBase: http://www.wormbase.org/). The Ppa-R144.2b-like gene is SL1 spliced, suggesting that it represents the most upstream gene in this P. pacificus operon. Taken together, these results suggest that P. pacificus contains operons and trans-splicing to SL2-like leaders, but the genes that are organized in operons are different between both species. Also, we could not obtain any evidence that a putative Ppa-ced-4 homolog would be part of this operon.

SL Specificity of the Ppa-ar-1 Operon
To test the SL specificity of the three genes in this operon, RT-PCR experiments were performed (fig. 2F). In C. elegans, it has been well established that SL2 spliced genes do also accept SL1 splicing (Blumenthal 1995; Blumenthal et al. 2002). We made similar observations for the obtained operon in P. pacificus. The Ppa-R144.2b-like gene is exclusively SL1 spliced, an observation that fits well with the fact that it is the most upstream gene in the operon. In contrast, both of the downstream genes are spliced to SL1 as well as to Ppa-SL2a and Ppa-SL2b. Thus, as in C. elegans, SL2 spliced genes do also accept SL1 splicing in P. pacificus.

Cloning of the P. pacificus SL1 RNA Genes
In C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1, the spliced leaders SL1 and SL2 are donated by small nuclear RNAs of around 100 nt in length. However, SL1 and SL2 gene organization differs from one another and differs between species. In C. elegans, the SL1 RNAs are encoded by about 110 genes, which are located in the same tandem repeat that specifies the 5S rRNA (Krause and Hirsh 1987). In contrast, the C. elegans SL2 RNAs are encoded by more than 30 unlinked genes (Huang and Hirsh 1989). In Oscheius/Dolichorhabditis sp.1 CEW1, the SL1 genes are not clustered and there is no indication for linkage with the 5S rRNA (fig. 1) (Evans et al. 1997).

To clone the P. pacificus SL1 genes, the P. pacificus Hind III BAC library was screened with a labeled oligonucleotide complementary to the first 21 nt of the P. pacificus SL1 spliced leader. The Ppa-SL1 fragment hybridized to 10 BAC clones, six of which have similar BAC end sequences (data not shown). Analyzing one of the BAC clones, PPBAC-21E22, in more detail revealed that the SL1 genes are organized as tandem repeats. Specifically, SL1 genes are approximately 100 nt in length, and neighboring SL1 genes are separated by around 500 nt (fig. 3A). All sequenced subclones have identical SL1 gene sequences and similar, but somewhat different, flanking regions. In contrast to C. elegans, the 5SrRNA gene is not present in the same tandem repeat as the SL1 genes in P. pacificus (fig. 3A). We were unable to find 5S rRNA sequences in any of the sequenced subclones of PPBAC-21E22. At the same time, analysis of the more than 20,000 available BAC end sequences of P. pacificus revealed the presence of 5S rRNA genes sequences in seven BAC clones, none of which hybridized with the SL1-specific probe.



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FIG. 3. SL genes and sequences from P. pacificus, C. elegans, and Oscheius (Dolichorhabditis) CEW1. (A) DNA sequence of one subclone of PPBAC-21E22 with two SL1 genes in an approximately 900-bp fragment. The sequence corresponding to the final SL1 spliced leader (SL1) and the rest of the SL1 gene (SL1 RNA gene) are shown separately with arrows. The two SL1 genes of this clone are separated by approximately 500 nt and are to a large part identical. (B) Sequence comparison of the SL1 genes between P. pacificus, C. elegans, and Oscheius (Dolichorhabditis) CEW1. Surprisingly, the SL1 gene of P. pacificus and C. elegans are more similar to one another than the one of the phylogenetically more closely related species C. elegans and Oscheius (Dolichorhabditis) CEW1. For P. pacificus and C. elegans, the consensus sequence is given. (C) Sequence comparison of various SL2-like genes between P. pacificus, C. elegans, and Oscheius (Dolichorhabditis) CEW1. Only the SL2b and SL2c genes were cloned genomically

 
Sequence comparison of the SL1 genes of P. pacificus, C. elegans, and Oscheius/Dolichorhabditis sp.1 CEW1 shows similarity patterns that are not consistent with the phylogeny of these three species. The Ppa-SL1 genes have an SL1 leader sequence that is identical to the one of the two other species and is followed by approximately 70 nt that could be folded into the canonical SL secondary structure. Surprisingly, these 70 nt are more similar between P. pacificus and C. elegans than between C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1 (fig. 3B). Specifically, there is a 61% sequence identity between P. pacificus and C. elegans but only of 31% sequence identity between C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1. Taken together, the SL1 gene organization in P. pacificus shows similarities and differences to C. elegans. Like the Cel-SL1 genes, Ppa-SL1 genes are organized in a tandem repeat, whereas the 5S rRNA gene is not linked to Ppa-SL1, a feature that P. pacificus shares with Oscheius/Dolichorhabditis sp.1 CEW1.

Cloning of the P. pacificus SL2b RNA Gene
To clone the P. pacificus SL2b gene, we applied a strategy recently developed by Siebert et al. (1995). Genomic DNA was digested with restriction enzymes and subsequently ligated to specific adaptors. We used a specific PCR primer designed against the 21 nt of the spliced leader and performed PCR experiments with an adaptor-specific primer. The resulting PCR fragments were cloned and sequenced and were then used to screen the P. pacificus Hind III BAC library. The Ppa-SL2b fragment hybridized to seven clones of the Hind III BAC library located in contig 295 of the physical map (Srinivasan et al. 2003). The Ppa-SL2b gene has a slightly different sequence than the known SL2-like sequences from C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1, but it can form a three-stem-loop secondary structure (figs. 3C and 4). Specifically, the first and second stems show little or no variation between the genes and the different species, whereas the third stem shows several variable positions between genes and species.



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FIG. 4. Comparison of proposed secondary structures of SL1 and SL2 RNAs from C. elegans and P. pacificus. For C. elegans, the SL2a RNA and for P. pacificus, the SL2b RNA are shown. Changes in the C. elegans SL2b RNA and the P. pacificus SL2c RNA are shown in italics. A {Delta} represents a gap in the alignment. The C. elegans data are according to Evans et al. (1997)

 
The PCR with the Ppa-SL2b leader sequence (Siebert et al. 1995) also resulted in the identification of another SL2-like gene of P. pacificus that has been named Ppa-SL2c (fig. 3C). Ppa-SL2c can also form a three-stem-loop secondary structure, and Ppa-SL2b and Ppa-SL2c differ in only 15% of the sequence. These results suggest that P. pacificus contains several SL2-like genes, a phenomenon that is similar to C. elegans and Oscheius/Dolichorhabditis sp.1 CEW1. In general, SL2-like genes seem to be more similar to one another within one species than between species. However, this information has to be considered with care as long as not all SL2-like genes are known from P. pacificus and Oscheius/ Dolichorhabditis sp.1 CEW1.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We studied polycistronic messenger organization and trans-splicing in Pristionchus pacificus and provided the first evidence for the existence of operons in free-living nematodes outside of the Rhabditidae. We identified an operon, trans-splicing to SL2-like leaders, the existence of several SL2-like leader sequences in the genome, and the clustering of SL1 RNA genes in tandem repeats, all features that have previously been described for C. elegans and partially also for Oscheius/Dolichorhabditis sp.1 CEW1. All of these features together strongly support the existence of operons in P. pacificus. It should be noted however, that we have been unable to obtain polycistronic cDNA clones, a phenomenon also known from C. elegans. Most likely, trans-splicing occurs cotranscriptionally, so the downstream genes in operons are only transcribed when the mRNA of the upstream gene has already been processed.

Evolutionary Implications for Operons in Nematodes
Although the existence of trans-splicing to SL1 is well documented for nematodes other than C. elegans, operons and SL2 splicing were previously only detected for species of the genus Caenorhabditis and Oscheius/Dolichorhabditis sp.1 CEW1 (Evans et al. 1997). The observations described here for P. pacificus suggest that operons and SL2 splicing are characters shared by the last common ancestor of the families Diplogastridae and Rhabditidae, which lived approximately 100 to 200 MYA (fig. 1).

At the same time, there is no evidence that genes organized in operons in C. elegans are also organized in operons in P. pacificus. Several genes that are organized in operons in C. elegans seem to be presented by monocistronic RNAs in P. pacificus, such as Ppa-cyt-1. In the case of the aldose reductase C35D10.6, the gene is organized in an operon in both species. However, the other genes in the "aldose reductase" operon in P. pacificus and C. elegans are not homologous to one another; that is, we could not identify a Ppa-ced-4-like gene and the other members of CEOP3224 in this operon (fig. 2A and B). Therefore, it remains unknown whether the last common ancestor of the Diplogastridae and Rhabditidae already contained an "aldose reductase" operon that would be similar to what is seen in any of the two present-day species. As none of the genes seems to be functionally related, the case of the "aldose reductase" operon does not provide any evidence for a functional relationship of genes found in the same operon.

Unexpected SL1 Gene Organization and Sequences Among Nematodes
SL1 gene organization and sequences vary substantially between P. pacificus, C. elegans, and Oscheius/Dolichorhabditis sp.1 CEW1 in a way that is not consistent with the phylogenetic relationship of these three species. We speculate that the tandem organization as observed in P. pacificus and C. elegans represents an ancestral state of SL1 gene organization and that the single gene organization known from Oscheius/Dolichorhabditis sp.1 CEW1 indicates a derived character. This hypothesis is also consistent with the surprisingly low sequence similarity of the SL1 genes (not, however, the final SL1 spliced leader) from Oscheius/Dolichorhabditis sp.1 CEW1 with the ones from P. pacificus and C. elegans. One can speculate that gene conversion–like mechanisms are involved in keeping the multiple copies of the SL1 genes identical within the tandem repeats in P. pacificus and C. elegans. Gene conversion has been proposed as a special recombination mechanism that is involved in keeping multiple copies of the same gene identical within one organism, such as for the ribosomal genes of most eukaryotes. In contrast, no such mechanism would operate in Oscheius/Dolichorhabditis sp.1 CEW1 after the SL1 genes lost their tandem organization. The absence of such mechanisms could also be involved in the acquisition of additional sequence changes that result in the unexpected observation that the Oscheius/Dolichorhabditis sp.1 CEW1 SL1 gene sequences are less similar to C. elegans than the ones from P. pacificus.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Alexandra Brand for technical assistance and Ray Hong for comments on the manuscript.


    Footnotes
 
Kenneth H. Wolfe, Associate Editor Back


    Literature Cited
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 

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Accepted for publication July 31, 2003.