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Journal of Experimental Botany, Vol. 56, No. 409, pp. 91–99, January 2005 doi:10.1093/jxb/eri014 Advance Access publication 8 November, 2004 This paper is available online free of all access charges (see http://jxb.oupjournals.org/open_access.html for further details) RESEARCH PAPER Analysis of two alleles of the urease gene from potato: polymorphisms, expression, and extensive alternative splicing of the corresponding mRNA Claus-Peter Witte*, Sarah Tiller, Edwige Isidore, Howard V. Davies and Mark A. Taylor† Scottish Crop Research Institute, Unit of Plant Biochemistry, Invergowrie, Dundee DD2 5DA, UK Received 12 May 2004; Accepted 20 August 2004 Abstract Introduction Globally, urea is the most widely used nitrogen fertilizer and is made accessible to plants via the urease reaction. However, sequence information for the plant enzyme is scarce. A cDNA encoding urease from soybean (Glycine max) has been cloned and sequence information has been obtained for two alleles (11 and 19 kbp, respectively) of the potato (Solanum tuberosum, cv. Désirée) urease gene and the corresponding cDNAs. It was found that urease is encoded by a single copy gene in several solanaceous species, and maps to chromosome V in potato. By contrast, the presence of two urease genes was reported for soybean. Comparative analysis of 11 kbp overlapping allelic DNA allowed the quantification of single nucleotide polymorphisms and revealed the presence of a truncated Ty1-copia retrotransposon in one of the alleles. Both alleles contained a copy of a terminal-repeat retrotransposon in miniature (TRIM). 25–50% of urease pre-mRNAs from both alleles were alternatively spliced in a variety of different ways. The retrotransposons had no effect on splicing. While urease is expressed in all tissues tested, its mRNA and protein are of low abundance. The TATA-less urease promoter appears to drive low-level housekeeping transcription. An in silico analysis showed that eukaryotic urease protein sequences are very similar to sequences from prokaryotic enzymes, conserving all residues of known functional importance. It is therefore likely that all known ureases are structurally similar, employing the same catalytic mechanism. Urease catalyses the hydrolysis of urea, yielding ammonia and carbonic acid. The enzyme is ubiquitous in plants (Frankenberger and Tabatabai, 1982; Hogan et al., 1983; Witte and Medina-Escobar, 2001) and is also found in bacteria, eukaryotic micro-organisms, and some invertebrates (Mobley and Hausinger, 1989). For plants, the urease reaction is essential to make urea-nitrogen accessible and urease deficiency can lead to urea accumulation in plant tissues (Shimada and Ando, 1980; Eskew et al., 1983). Urease is an enzyme of great agronomic importance, since most nitrogen fertilizer used worldwide is applied as urea (FAO, 1998). At the physiological level, urease has been studied in soybean (Polacco and Holland, 1993). Soybean contains a ubiquitous urease, expressed in all tissues, and an embryo-specific urease, accumulating to activity-levels 1000-times greater than those of ubiquitous urease. Two distinct genes encode these ureases (Torisky et al., 1994). The active site architecture of urease has been elucidated in Klebsiella aerogenes. Several histidines and an aspartate are ligands for two active site nickel ions (essential for activity; Dixon et al., 1975; Witte et al., 2002a) that, in addition, are bridged by a carboxyl group covalently attached to the terminal amino group of a lysine residue (Jabri et al., 1995). The incorporation of nickel into the active site of urease is aided by several urease accessory proteins in bacteria and plants (Freyermuth et al., 2000; Witte et al., 2001a; Bacanamwo et al., 2002). The seed enzyme from Canavalia ensiformis (jackbean) has long been the only eukaryotic urease for which full-length coding sequence information was available. The genomic sequences of three fungal enzymes (Schizosaccharomyces Key words: Adenylate kinase, alternative splicing, SNP, TRIM, Ty1-copia, urease. * Present address: Max-Planck-Institute for Plant Breeding Research, Department of Plant Microbe Interactions, Carl-von-Linné-Weg 10, D-50829 Cologne, Germany. y To whom correspondence should be addressed. Fax: +44 (0)1382 562426. E-mail: [email protected] Journal of Experimental Botany, Vol. 56, No. 409, ª Society for Experimental Biology 2004; all rights reserved 92 Witte et al. pombe, Cryptococcus neoformans, Coccidioides immitis) and of a second plant enzyme (from Arabidopsis thaliana) have now been published. Very recently, the coding sequences for the ubiquitous and the seed-specific ureases of soybean (Glycine max) have been added to the database (Goldraij et al., 2003). Ureases have been cloned from two further plant species: a cDNA from Glycine max (soybean) and two alleles of the urease gene from Solanum tuberosum (potato). In potato, as well as in several other solanaceous species, urease is shown to be a single copy gene. The two alleles of the potato urease gene were analysed for polymorphisms, and extensive alternative splicing of the corresponding mRNAs in leaf and root tissues was discovered. It was demonstrated that urease is expressed ubiquitously in the potato plant and an in silico analysis of urease sequences highlights the similarity of prokaryotic and eukaryotic enzymes. Materials and methods DNA and RNA isolations Genomic DNA was obtained as previously described (Draper et al., 1988). RNA was extracted from greenhouse-grown plants (soybean leaves or potato cv. Désirée leaves) using the TRI reagent (Sigma) following the manufacturer’s protocol. For analysis of differential splicing, leaves (4th–5th node from the apical shoot tip) and roots from young potato plants before the flowering stage were used. Isolation of potato urease genomic clones A lambda EMBL-3 SP6/T7 phage library of the potato genome (cv. Désirée; Clontech, Palo Alto, CA) was screened twice following the manufacturer’s protocol. For the first screen, probe DNA (320 bp) was generated by RT-PCR (total leaf RNA, RT-step with Superscript II from Gibco BRL, 41 8C annealing temperature, 40 cycles) with degenerate primers: GGITTIAARITICAYGARGAYTGGG (forward) and TGRTGRCAIACCATIARCATRTC (reverse). This yielded two clones, k1 and k2b, representing different alleles of the Désirée urease gene, but which lacked the 59-end of the gene. Therefore, a second screen was performed using a probe matching the 59-end of the gene. This probe was generated as follows. In a first step, a degenerate primer matching the 59-coding sequence of jackbean and soybean urease ATGAARYTIWSICCIAGRGA (for) and two reverse primers AGGGAATTACATTCTTTACACCACA (rev1) CTGAATTGGTCTGTCACCTG (rev2) were used in nested RT-PCR (total leaf RNA; RT-step using rev1 with Superscript from Gibco BRL; 20 cycles during the first PCR with 48 8C annealing temperature using for and rev1; 25 cycles during nested PCR with 48 8C annealing temperature using for and rev2. Based on the sequence of the obtained PCR product, a third reverse primer CAGGAAGGAAAGAACCAT (rev3) was designed to allow the amplification of a probe fragment not overlapping with the previously isolated lambda phages (PCR with primers for and rev3; 20 cycles, 48 8C annealing temperature). This probe identified a single clone (k2a) which contained the 59 end of the potato urease gene and overlapped with k1 and k2b. Isolation of potato urease cDNA Based on the genomic sequence, primers were designed to the 59 and 39 end of the coding sequence: CATATGAAGTTGGCTCCAAG (59) and TAGGAATTCCTAAAAGAGGAAATAGTTCCTGGAG (39). A reverse transcription step using primer 39 was carried out with MMLV-reverse transcriptase from Gibco BRL (3 lg total RNA from root or leaf as template) as described by the manufacturer. Subsequent PCR was performed with forward and reverse primers for 32 cycles (53 8C annealing temperature, 68 8C elongation temperature, Roche HiFidelity Taq DNA Polymerase). Resulting amplicons (2.5 kbp) were purified from an agarose gel and cloned into pGEM-T Easy (Promega). 10 clones from leaf and 12 clones from the roots were sequenced. The coding sequence of potato urease was predicted from the genomic sequence by alignment with the jackbean urease cDNA. This prediction was confirmed by aligning the potato cDNA sequences, which also revealed the occurrence of alternative splicing. Isolation of soybean urease cDNA A degenerate primer was designed based on the published N-terminal amino acid sequence of ubiquitous soybean urease (Torisky et al., 1994): ATGAARYTIWSICCIAGRGA (for). Published partial sequence of ubiquitous soybean urease (accession number EMBL: S69179) was used to design a primer to the 39-end: CTTAAAAGAGGAAGTAATTTCGAG. Total RNA from soybean leaf (1.2 lg) was reverse transcribed with Superscript reverse transcriptase from Gibco BRL. 30 cycles of PCR (50 8C annealing temperature) with both of the above primers amplified a 2.5 kbp product which was sequenced. RACE-PCR (see below) was used to obtain non-degenerate 59-end sequence. A second RT-PCR experiment (conditions as before) using primer ATTTCTGAATTCTCTGCTGC (which binds upstream of the start codon) and the previously used reverse primer was conducted in triplicate. Possible PCR errors were detected by comparing sequences of three clones obtained from independent RT-PCR reactions. RACE-PCR 59 RACE-PCR was conducted following the instructions of the Roche 59/39 RACE-PCR kit, using Superscript reverse transcriptase and terminal transferase from Gibco BRL. For 59-RACE of soybean urease, the following gene-specific primers were designed: TGACTAACTTAGTACCATCTCG (for reverse transcription and first PCR; 16 cycles, 54 8C annealing temperature) and GATGAGGAACAGYTGGAAGAACTTG (for nested PCR; 30 cycles, 60 8C annealing temperature). For potato urease 59-RACE, the primers rev1, rev2, and rev3 described previously were used. The reverse transcription step was carried out with primer rev1, primer rev2 was used in a first PCR (20 cycles; 55 8C annealing temperature), and primer rev3 in a nested PCR (30 cycles; 61 8C annealing temperature). Southern analysis Southern analysis was carried out with DNA from several potato cultivars: Désirée, Stirling, Gladstone, Lumpers, Teena; different Solanum species (accession numbers in the Commonwealth Potato Collection at the Scottish Crop Research Institute, Dundee, in brackets): S. phureja (IVP 48), S. acaule ssp. aemulans (ACL 7016), S. brachycarpum (BCP 7027), S. chacoense ssp. chacoense (CHC7143), S. papita (PTA 7079), S. ochranthum (OCR 7517); and three further solanaceous species: Lycopersicon esculentum (cv. Moneymaker), Nicotiana benthamiana, and Datura stramonium. Approximately 30 lg of either EcoRI or HindIII-digested DNA was loaded per lane. The blots were probed with a 215 bp fragment of exon 18 of the potato urease gene, generated by PCR using the reverse primer (39) described previously and a forward primer: TGGATGCTGGAATAAAAG. Washes were performed at 65 8C with 23 SSC (saline sodium citrate buffer), 0.1% SDS (sodium dodecyl sulphate; twice for 5 min) and 0.13 SSC, 0.1% SDS (twice for 15 min). The washed blots were exposed to film for 22 h. Mapping of potato urease A potato diploid F1 population (SH83-92-488 3 RH89-039-16; Rouppe van der Voort et al., 1997) was used to determine the map location of urease. Two primers AGGCTGCAAGTTCTAATTC and Analysis of two alleles of the potato urease gene 93 GTGTATACAAAGTAAATCCCAAC were used to amplify a simple sequence repeat region in intron 17. Reaction conditions, separation, and visualization of reaction products were as described in Provan et al. (1996). Genetic linkage analysis was performed using Joinmap version 2.0 software (Stam and van Oijen, 1995). Protein extraction, electrophoresis, and in-gel urease detection Tissue from field-grown potato plants before the flowering stage were used for protein extraction in 50 mM phosphate buffer (pH 7.5) containing 1.5% PVPP, 50 mM NaCl, 1 mM EDTA, 10 mM DTT, and 0.1 mM PMSF (tissue to buffer ratio=1:3). Extracts were centrifuged at 20 000 g for 20 min at 4 8C and protein concentrations of the supernatants were determined by a commercial Bradford assay (Bio-Rad) using BSA as standard. Electrophoresis and in-gel staining were performed as described by Witte and Medina-Escobar (2001). 75 lg protein were loaded per sample (except for the mother tuber, because of low protein concentration: loading 13 lg). Bioinformatic tools Sequence alignments of ureases were generated with Clustal W (Thompson et al., 1994) using default settings. The three urease subunit sequences of Klebsiella aerogenes were fed in as a single contiguous sequence. Shadings were generated using the public domain program BOXSHADE. Urease and adenylate kinase sequences were identified by BLAST searches (Altschul et al., 1997). Single nucleotide exchanges and Indels were identified by pairwise alignment of the genomic urease clones using the GAP program (University of Wisconsin Genetics Computer Group). TRIM elements were discovered as described by Witte et al. (2001b). Results Cloning and structural analysis of the potato urease gene Three overlapping lambda clones (k1, k2a, k2b) containing the urease gene sequence of Solanum tuberosum ssp. tuberosum (cv. Désirée) were isolated from a lambda phage genomic DNA library (Clontech; Fig. 1). Fragments k2a and k2b were identical in the overlapping region and the joined sequence (18 962 bp; accession number EMBL: AJ276864; subsequently referred to as k2) contains one allele of a full-length urease gene, comprizing 18 exons (Fig. 1; exons 1–18). Three additional possible exons were found upstream of the urease gene, coding for a peptide with strong similarity to the C-terminal sequence of plant adenylate kinases (Fig. 1; exons A–C). The fragment k1 (11 066 bp, accession number EMBL: AJ276865) overlaps completely with k2 (Fig. 1), but is not identical to it. The most obvious difference is an insertion of 1755 bp in intron 6 of k2 absent in k1 (Fig. 1; insertion). This insertion represents a retrotransposon, containing long terminal repeats of 261 bp, but lacking internal coding regions for mobility factors, indicating that this element is not capable of autonomous transposition. Some fragmented remnants of coding sequence for reverse transcriptase and RNaseH allowed the element to be classified as Ty1-copia. The Ty1-copia element is flanked by further repeats belonging to a terminal-repeats retrotransposon in miniature (TRIM; Fig. 1). The TRIM is also present at the same position in k1, but is not interrupted as in k2. The nonautonomous TRIM-type of retrotransposons were first discovered in the potato urease gene, but were later found to be widespread in genomes of monocot and dicot plant species (Witte et al., 2001b). Urease is a single copy gene in potato and related species Whether k1 and k2 are two alleles of the same gene or represent different genes was tested by Southern Blot and mapping analysis. Using a fragment of exon 18 as probe, single hybridizing fragments were observed in Southern blots generated with EcoRI and HindIII digested genomic DNA of several cultivars of Solanum tuberosum and various other solanaceous species, including tomato, Datura stramonium, and Physalis floridana (Fig. 2). The restriction fragments expected for the potato cv. Desireé are marked in the genomic map of the urease gene (Fig. 1). For N. benthamiana a faint band could only be observed after prolonged exposure (not shown). These data indicate that these species possess a single copy gene for urease, whereas two urease gene copies were reported in soybean (Torisky Fig. 1. Schematic alignment of genomic urease clones from potato (drawn to scale). Exons are shown as dark shaded boxes. Adenylate kinase exons are labelled with capital letters, urease exons are numbered (above the alignment). The Ty1-copia element is marked with a grey shaded box, the TRIM element with an open box containing a diagonally crossing line. EcoRI and HindIII fragments seen on the Southern blots (Fig. 2) are marked, the downstream restriction site for the HindIII fragment is unknown. 94 Witte et al. 1999). Thus, it is likely that urease is a single copy multiallelic gene in potato and probably also in several other solanaceous species. SNPs and Indels between the two allelic urease copies Genomic subclones of the urease gene alleles were sequenced on both strands and most regions on each strand was sequenced more than once. A detailed comparison of the overlapping region between the two urease alleles revealed the presence of 277 single nucleotide polymorphisms (SNPs; on average, 2.5 polymorphisms per 100 bp: 2.5%). For non-coding regions a SNP frequency of 2.8% was found while in coding regions the frequency was 1.2%. Within the non-coding regions 40 insertion/deletion (Indel) events can be documented. Of these 70% are 1–4 bp in length, 20% are 5–10 bp, and 10% (four events) are longer than 10 bp. Possible causes for most of the longest Indels may be deduced from sequence features. As mentioned above, the insertion in intron 6 (k2) is due to a retrotransposon; an Indel of 30 bp is found in an array of 30 bp repeats in intron 14 which is probably caused by unequal cross-over; and an Indel of 34 bp in intron 17 is present in a SSR region, which are known to undergo expansion and contraction probably due to polymerase slippage during replication (Schlötterer and Tautz, 1992). The urease promoter and urease expression Fig. 2. Autoradiography of two Southern blots containing EcoRIdigested (a) or HindIII-digested (b) DNA from different solanaceous species, probed with a fragment of exon 18 of potato urease. The first five lanes contain DNA from different potato cultivars, the next six lanes contain DNA from wild-type Solanum species, the last three lanes contain DNA from solanaceous species. See Materials and methods for species accession numbers and experimental details. et al., 1994). The presence of a second urease copy could not be ruled out for Solanum papita, Solanum acaule, and Solanum brachycarpum because two hybridizing bands were observed when genomic DNA was digested with HindIII (for S. acaule also with EcoRI; Fig. 2). A simple sequence repeat (SSR) present in intron 17 of the sequenced urease gene from Solanum tuberosum (cv. Désirée) was used to map the gene on an ultra-high density AFLP (amplified fragment length polymorphism) map (Isidore et al., 2003). The gene mapped to a single location on chromosome 5, near Gp188 and Stm1020 (Collins et al., The urease transcription start site was localized by 59-RACE at ÿ60 (translation start site= +1). Although a canonical TATA element and other typical promoter elements cannot be found by manual inspection, computational promoter prediction using a neural network (server at http://www. fruitfly.org/seq_tools/promoter.html; Reese and Eeckman, 1995) locates a possible transcription start site at ÿ57, indicating the presence of possible promoter elements. The absence of a canonical TATA box has been reported for other genes (Dynan, 1986; Ibrahim et al., 2001). Steady-state levels of urease mRNA were found to be undetectable by northern blot analysis using 30 lg of total RNA from a variety of organs (not shown), suggesting either a low level of transcription or instability of urease mRNA. In western blots using a polyclonal antibody against jackbean seed urease, the potato enzyme could be detected in all organs investigated, but only close to the detection limit (not shown). In-gel urease activity staining showed that urease activity is ubiquitously present in the potato plant (Fig. 3) which is consistent with results obtained from activity measurements in different tissues (Witte et al., 2002b). Thus, the urease promoter appears to drive low-level housekeeping expression of the urease gene. However, urease activity levels seem more than sufficient for the plant’s needs since urea applied to the leaves is hydrolysed more rapidly than the resulting ammonium can be assimilated (Witte et al., 2002b). Analysis of two alleles of the potato urease gene 95 Fig. 3. In-gel urease activity stain for different potato organ extracts. Sink leaves are leaves from the second node under the apical shoot tip, source leaves are leaves directly above the ground. Purified jackbean urease (8 mU; Boehringer) was loaded as positive control (last lane). Secondary bands stem from different aggregation states of urease (Fishbein, 1969). Comparative analysis of ureases from different eukaryotes Coding sequences of both allelic urease forms from potato and of ubiquitous urease from soybean were compared to all other eukaryotic urease sequences available (two plant and three fungal sequences) and also to 36 full-length bacterial sequences. An alignment focusing on the plant sequences compared with the sequence from Schizosaccharomyces pombe and Klebsiella aerogenes is shown in Fig. 4. In most bacteria, urease is encoded by three genes (ureA, ureB, and ureC). Eukaryotic urease genes contain regions of similarity collinear to all three bacterial genes, bridged by short linking regions not present in bacteria (ureA-bridge-ureB-bridge-ureC). The degree of sequence conservation among ureases of different origin is high. For example, ureA, ureB, and ureC from K. aerogenes are 54%, 50%, and 61% identical at the protein level to the corresponding sequences of potato urease (k2), while urease from A. thaliana and potato (k2) are 75% identical. The cDNA sequence of the ubiquitous soybean urease that was recently deposited in the database (accession number EMBL: AY230156) is identical to the sequence obtained in this study except for one silent nucleotide exchange. Detailed biochemical studies have been carried out on urease from Klebsiella aerogenes. All residues biochemically shown, or structurally inferred, to be important for optimal enzyme performance in K. aerogenes are conserved in eukaryotic sequences (Fig. 4; amino acid labelling according to K. aerogenes urease). This includes the residues that co-ordinate nickel in the active site of K. aerogenes urease (Hisa134, Hisa136, Hisa246, Hisa272, and Aspa360) and the lysine residue carrying the carboxyl group which bridges the two active site nickel ions (Ka217; Park and Hausinger, 1993; Jabri et al., 1995). It also includes Hisa219, thought to aid in the polarization of the urea carbonyl group, and Hisa320, thought to act as a general acid during catalysis, as well as Da221 and Ra336, implied in the modulation of Hisa320 orientation and acidity (Pearson et al., 2000). Finally, it includes Cysa319, shown not to be essential for catalytic activity but to facilitate catalysis at neutral and basic pH values (Martin and Hausinger, 1992; Pearson et al., 1997). Given the high degree of overall conservation and the conservation of catalytically important residues among prokaryotic ureases, and also between eukaryotic and prokaryotic enzymes, it seems likely that all ureases described to date are structurally similar, employing the same catalytic mechanism. The crystal structure of K. aerogenes urease is consistent with this notion, since the bridging regions that connect ureA, ureB, and ureC in eukaryotes could be accommodated without significantly altering the structure (Jabri et al., 1995). Alternative splicing of potato urease The insertion of retrotransposons into an intron of a gene has the potential to alter pre-mRNA splicing patterns (Marillonnet and Wessler, 1997). To investigate whether the presence of the Ty1-copia and retrotransposon in intron 6 of the k2-urease allele hinders the generation of functional mRNA from this allele, urease-specific mRNA was amplified by RT-PCR from leaf and root tissues, with primers flanking the complete coding region. Ten clones from leaf and 12 clones from root were sequenced to test the integrity of the coding sequence (accession number EMBL: AJ308543 and EMBL: AJ308544 for allelic forms 1 and 2, respectively). Surprisingly, urease-specific mRNA was found to be differentially spliced at various locations (Table 1; Fig. 5), but splicing of the retrotransposoncontaining intron was accurate in both alleles, indicating that the transposon insertion does not influence splicing efficiency of this intron. In the leaf, four of the 10 sequenced clones showed alternative splicing compared with expected splicing, whereas in the root, alternative splicing was found in two out of 12 clones, with one clone being affected by two different events (Table 1). Retention of intron 11 was the only alternative splicing event that could be documented twice (once in the leaf and once in the root), all other cases differed from each other, affected introns 4, 5, 7, 9, 10, 11, 12, and 17 indicating widespread changes in splice site choice throughout the gene (Table 1). In several cases, a small shift in either the 39 or the 59 splice site had occurred, but more extensive mRNA alterations were also observed, including the skipping of exon 10, the retention of intron 11, and the inclusion of 73 bp of intron 4 as an exon (Fig. 5). In all but one case (Table 1; line 1), alternative splicing introduced stop codons, shortening the open reading frame. Urease amplicons were size-selected before cloning (see Materials and methods), thus putative alternative splicing events that would greatly alter mRNA size were not selected. It can therefore not be excluded that such splicing events occur. Discussion Gene copy number In contrast to the situation in soybean, urease is a single copy gene in potato and several related solanaceous species 96 Witte et al. Fig. 4. Alignment of ureases, generated with Clustal W using default parameters. S. tub., Solanum tuberosum (allele k2); sequence variations in k1 are given above the alignment; G. max, Glycine max; C. ens., Canavalia ensiformis (Swissprot: p07374); A. tha., Arabidopsis thaliana (SpTrembl: aaf07806); S. pom., Schizosaccharomyces pombe (SpTrembl: cab52575); K. aer., Klebsiella aerogenes (Swissprot: p18314; p18315; p18316 for subunits A, B, and C, respectively). Arrows underneath the alignment show the approximate intron positions in ureases from S. tuberosum and A. thaliana. The arrow points to the amino acid whose codon is at least partially part of the preceding exon. Residues shown to be important for urease function in K. aerogenes are marked with black triangles, numbered with the amino acid position in the corresponding K. aerogenes subunit. Residues tested, but not found to be important for function are marked with an asterisk. Analysis of two alleles of the potato urease gene 97 (Fig. 2). The genome of A. thaliana also contains only one copy of urease. A second copy of urease in soybean (and related species) was probably the result of a gene duplication event, allowing the development of an enzyme expressed mainly in seed. While the ubiquitous urease enzyme, found in all organs in soybean, is essential for the recycling of metabolically derived urea (Stebbins et al., 1991), the function of the seed-specific enzyme is less clear (Polacco and Holland, 1993). In potato, urease activity was detected in all organs investigated (Fig. 3; Witte et al., 2002b). The potato urease gene is therefore likely to be the functional equivalent to the ubiquitous urease gene of soybean. SNPs and Indels The comparison of 11 kbp of allelic DNA allowed the frequency of SNPs and Indels to be assessed in this region of the potato genome. The average frequency of 1 SNP every 40 bp between the two urease alleles is comparatively high. When 10 different loci from three Arabidopsis ecotypes were compared, polymorphism was detected only every 207–413 bp on average, although a greatly increased frequency was observed at one hypervariable locus (Hauser et al., 1998). An analysis of 39-untranslated regions of 20 loci in 30 maize lines revealed the presence of one SNP Table 1. Alternative splicing of potato urease mRNA Organ Allele Comments Leaf 1 Leaf 1 Leaf Leaf Leaf 1 1 2 Root 1 Root 2 Root 2 39 splice site of intron 9: last 9 bp of intron 9 included in exon 10; a GG codon was chosen as the 39-splice site 39 splice site of intron 17: first 4 bp of exon 18 spliced out with intron 17 exon 10 skipped intron 11 retained 73 bp internal sequence of intron 4 included as an exon (position: 6270–6342 in EMBL: aj276864) (a) 39 splice site of intron 7: first 5 bp of exon 8 spliced out with intron 7 (b) intron 11 retained 59 splice site of intron 5: last 17 bp of exon 5 spliced out with intron 5 39 splice site of intron 12: first 5 bp of exon 13 spliced out with intron 12 every 70 bp and one Indel every 160 bp on average (Rafalski et al., 2001). In the human genome, SNPs occur once every 100–300 bp (SNP database at http://www. ncbi.nlm.nih.gov/SNP/). To gain a more comprehensive view of the general genetic variability in the potato genome, the analysis of several loci would be required. Given the high degree of polymorphism at the urease locus it is surprizing that generally single bands were observed in Southern blot analysis (for all autoploid Solanum species, Fig. 2). Multiple bands were only observed for the allotetraploid species S. papita and S. acaule and for the allohexaploid species S. brachycarpum. Alleles k1 and k2 are the only allelic forms occurring in the autotetraploid cultivars/species tested, or perhaps other additional alleles are considerably less polymorphic. Conservation of ureases Ureases are highly conserved across eukaryotic and prokaryotic kingdoms (Fig. 4). This analysis has shown that all the known important features characteristic of ureases in bacteria were preserved in plants (eukaryotes). The catalytic mechanism employed by plant (eukaryotic) ureases is therefore unlikely to vary significantly from what has been found for bacteria. The organization of the urease genes in bacterial operons is collinear with the regions of similarity to these genes found in eukaryotic ureases. The presence of merged ureA and ureB genes in the only distantly related bacterial groups Helicobacter and Deinococcus suggest that merging events creating one single reading frame (as found in eukaryotes) seem likely to have happened more than once during evolution. Alternative splicing Alternative splicing occurs through variable splice site selection, which results in the production of different mRNAs derived from the same gene and can give rise to distinct proteins. In humans, over 60% of genes are estimated to be alternatively spliced and show multiple splicing patterns (Black, 2003). By contrast, around 5% of Arabidopsis genes have been estimated to show alternative Fig. 5. Schematic drawing of some alternatively spliced urease mRNAs drawn to scale. Exons are given as alternate dark and light shaded boxes. Intronic sequence is marked by using white boxes. (a) Correctly spliced; (b) exon 10 skipped; (c) intron 11 retained; (d) part of intron 4 included as an exon into the mRNA (fragment of genomic DNA showing intron 4 is given above the mRNA). Arrows mark stop codons. 98 Witte et al. splicing of a type less complex than that described for vertebrates and invertebrates (Brett et al., 2002). Nevertheless, an increasing number of alternative splicing events have been described to have roles in plant metabolism and development (Tantikanjana et al., 1993; Comelli et al., 1999; Figueroa et al., 1999; Mano et al., 1999; Quesada et al., 2003). Alternative splicing also leads to a truncated coding sequence due to the activation of intron polyadenylation signals or by bringing translation stops in-frame. In some cases these proteins retain activity whereas in others, activity is lost. (Kyozuka et al., 1997; Nishihama et al., 1997; MacKnight et al., 1997). More recently, the presence of nonsense termination codons have been described to lead to mRNA degradation through nonsensemediated decay (Maquat, 2004). Such processes regulate the levels of relevant mRNA available for translation and, therefore, can regulate gene expression. Urease mRNA in potato is subject to extensive alternative splicing at different introns throughout the length of the gene’s pre-mRNA. It is likely that the number of differentially spliced pre-mRNAs is greater than that described here due to the limited number of clones sequenced. Recently, the presence of urease-like sequences in jackbean was reported (Pires-Alves et al., 2003) which may also be the result of differential splicing events. In most cases, alternative splicing found in potato urease mRNAs led to the introduction of stop codons giving rise to shortened ORFs that lack the catalytic motifs. Thus, aberrant splicing probably results in a reduction of urease protein, because a great proportion (50% in leaf and 25% in root) of pre-mRNA was processed incorrectly (Table 1). This inaccuracy (relaxed splicing) may be tolerated as long as sufficient intact urease mRNA is made. In leaves of potato, sufficient urease activity is present as the substrate, urea, is barely detectable. Even when urea is applied externally, urease is not rate-limiting for the assimilation of urea nitrogen (Witte et al., 2002b). Thus, despite the low level of expression and the relaxed splicing of urease pre-mRNA, urease activity appears to be abundant for the plant’s needs. Selective pressure for improved expression or more accurate splicing is probably low. Relaxed splicing might not only affect urease, but also other gene transcripts. While in some instances a functional role for an alternatively spliced transcript may emerge, in the majority of cases the resulting mRNA is likely to encode non-functional or at best equally functional proteins. The current literature on alternative splicing in plants is consistent with this notion. In cases where pre-mRNA processing is limiting for protein expression, splicing is probably very accurate due to selective pressure for strong splice signals. Conversely, a greater extent of relaxed splicing is likely to occur when protein amounts are more than sufficient or are limited by other factors. A certain tolerance/inaccuracy in splicing might be one element in the toolkit of evolution to aid the emergence of new functions from old genes. 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