Max-Planck Institut für Entwicklungsbiologie, Abteilung für Evolutionsbiologie, Tübingen, Germany
Correspondence: E-mail: ralf.sommer{at}tuebingen.mpg.de.
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: Pristionchus pacificus Caenorhabditis elegans operons trans-splicing aldose reductase
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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.
|
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.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 conversionlike 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bektesh, S., K. Van Doren, and D. Hirsh. 1988. Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes. Genes Dev. 2:1277-1283.[Abstract]
Blumenthal, T. 1995. Trans-splicing and polycistronic transcription in Caenorhabditis elegans. Trends Genet. 11:132-136.[CrossRef][ISI][Medline]
Blumenthal, T., D. Evans, and C. D. Link, et al. (11 co-authors). 2002. A global analysis of Caenorhabditis elegans operons. Nature 417:851-854.[CrossRef][ISI][Medline]
Eizinger, A., B. Jungblut, and R. J. Sommer. 1999. Evolutionary change in the functional specificity of genes. Trends Genet. 15:197-202.[CrossRef][ISI][Medline]
Evans, D., D. Zorio, M. MacMorris, C. E. Winter, K. Lea, and T. Blumenthal. 1997. Operons and SL2 trans-splicing exist in nematodes outside the genus Caenorhabditis. Proc. Natl. Acad. Sci. USA 94:9751-9756.
Fleischmann, R. D., M. D. Adams, and O. White, et al. (40 co-authors). 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269:496-512.[ISI][Medline]
Hengartner, M. O., and H. R. Horvitz. 1994. C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell 76:665-676.[ISI][Medline]
Huang, X. Y., and D. Hirsh. 1989. A second trans-spliced RNA leader sequence in the nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 86:8640-8644.[Abstract]
Jungblut, B., and R. J. Sommer. 1998. The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates. Curr. Biol. 8:775-778.[ISI][Medline]
Krause, M., and D. Hirsh. 1987. A trans-spliced leader sequence on actin mRNA in C. elegans. Cell 49:753-761.[ISI][Medline]
Maruyama, K., and S. Sugano. 1994. Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. Gene 138:171-174.[CrossRef][ISI][Medline]
McCarter, J. P., S. W. Clifton, D. M. Bird, and R. H. Waterston. 2002. Nematode gene sequences: update for June 2002. J. Nematol. 34:71-74.[ISI]
Sambrook, J., and D. W. Russell. 2001. Molecular cloning: a laboratorial manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.
Siebert, P. D., A. Chenchik, D. E. Kellogg, K. A. Lukyanov, and S. A. Lukyanov. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23:1087-1088.[ISI][Medline]
Sommer R. J., L. K. Carta, S.-Y. Kim, and P. W. Sternberg. 1996. Morphological, genetic and molecular description of Pristionchus pacificus sp. n. (Nematoda : Neodiplogastridae). Fundam. Appl. Nematol. 19:511-521.[ISI]
Sommer, R. J. 2000. Evolution of nematode development. Curr. Opin. Genet. Dev. 10:443-448.[CrossRef][ISI][Medline]
Spieth, J., G. Brooke, S. Kuersten, K. Lea, and T. Blumenthal. 1993. Operons in C. elegans: polycistronic mRNA precursors are processed by trans-splicing of SL2 to downstream coding regions. Cell 73:521-532.[ISI][Medline]
Srinivasan, J., W. Sinz, T. Jesse, L. Wiggers-Perebolte, K. Jansen, J. Buntjer, M. van der Meulen, and S. R. J.. 2003. An integrated physical and genetic map of the nematode Pristionchus pacificus. Mol. Gen. Genomics 269:715-722.[CrossRef][ISI][Medline]
Srinivasan, J., W. Sinz, and C. Lanz, et al. (15 co-authors). 2002. A bacterial artificial chromosome-based genetic linkage map of the nematode Pristionchus pacificus. Genetics 162:129-134.
Takacs, A. M., J. A. Denker, K. G. Perrine, P. A. Maroney, and T. W. Nilsen. 1988. A 22-nucleotide spliced leader sequence in the human parasitic nematode Brugia malayi is identical to the trans-spliced leader exon in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 85:7932-7936.[Abstract]