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1 Department of Biochemistry, Kawasaki Medical School, Kurashiki City, Okayama, Japan
2 Central Research Laboratory, Ajinomoto Co. Inc., Suzuki-cho, Kawasaki City, Japan
3 Molecular Biology Laboratory, Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi City, Chiba, Japan
4 Department of Human Genetics, University of Utah, Salt Lake City, UT and Biosciences Institute, University College, Cork, Ireland
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Abstract |
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Introduction |
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X174 function to inhibit peptidoglycan synthesis of the cell wall structures and subsequently induce host cell lysis (Bernhardt et al. 2000; Young et al. 2000; Bernhardt et al. 2001a, b). The RNA phage lysis product on its own is sufficient to trigger host lysis (Kastelein et al. 1982; Coleman et al. 1983; Karnik & Billeter 1983; Winter & Gold 1983). Although synthesis of phage MS2 lysis protein was reported to require a special ribosomal frameshifting event (Kastelein et al. 1982), later work showed that this is incorrect (Berkhout et al. 1987) and instead a proportion of ribosomes terminating coat protein synthesis scan ‘backwards’ (5') and initiate translation of the lysis gene (Adhin & van Duin 1990). However, the very different types of lysis proteins and contrasting gene locations between the group A and group B RNA phage, which are similar in other ways, is an enigma. We have studied a fortuitous frameshift mutant early in the replicase gene of Qß and find surprising lysis properties which provide evolutionary insights and hints of a possibly normal mechanism.
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Results |
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H1trp to construct a new plasmid/cell system to investigate the lysis. The derived plasmid pQßg-8 contains the same Qß sequence at the beginning of the replicase but is truncated after the stop codon which is brought into frame by the frameshift mutation. Cells containing this plasmid started to lyse very quickly on standard media (Fig. 4), whereas, with low salt concentration in LB media, they lysed gradually (Figs 4 and 5). This slow lowering of the growth profile of the host cells (pQßg-8) suggests that internal cell materials gradually leaked away through the cell wall. On induction, cells with plasmid, pQßA2-11, encoding the lysis/maturation protein A2, start to lyse slowly (Fig. 4). Cells with plasmid-encoded lysis genes derived from group A RNA phages behaved similarly (Nishihara 2002, 2003). These results suggest that Rg-lysis protein (encoded by pQß Rg-1 and pQßg-8) may lyse cells in a different way from that of QßA2-lysis proteins (encoded by pQßA2-11) and the lysis proteins of group A phages.
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Lysis of E. coli (K12H1trp) mediated by expression of plasmid pQßg-8 showed an unusual pattern (Fig. 4), prompting further analysis with different sodium chloride concentrations in LB medium. With sodium chloride in the media, induction of plasmid expression resulted in immediate lysis (Fig. 5) with the sharpest decrease in turbidity occurring with 4% sodium chloride (Fig. 6). In the control without sodium chloride, the growth curve remained flat for a long time (Fig. 5), and the cells were sensitive to EDTA, unlike control cells (Fig. 7). Addition of 1 mM EDTA caused a rapid decrease in turbidity indicating cell lysis, although simultaneous addition of magnesium ions neutralized the effect (Fig. 7). When 0.5 M sucrose was present and different concentrations of sodium chloride were added, plasmid expression resulted in cell turbidity increasing rapidly (Fig. 8), and the cells lysed in the same way as in the absence of sucrose (Fig. 5). With 0.5 M sucrose, absence of sodium chloride resulted in a corresponding rapid increase in turbidity, and the new level remained constant for the further 120 min examined (Fig. 8). In the same media, the rate of increase of control cells which lacked the lysis gene, was 1.2-fold.
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Transmission electron microscopy demonstrated that lysing E. coli cells (JM 105) expressing the mutant replicase gene (pQßRg-1), showed a wide variety of lysing shapes (Fig. 10). Ballooning structures, which seemed to be the outer membrane, were observed on the surface of cells (Fig. 10A,B). In Fig. 10(A), the ballooning structure seemed to be still intact, but in Fig. 10(B–F), cells were ruptured and cell materials were released. In Fig. 10(D), one cell was harshly ruptured. A similar observation was seen in other lysing cells by the cloned lysis gene of MS2 phage (Witte et al. 1989). Figure 11(A–C) shows the transmission electron micrographs obtained from lysing cells (K12H1trp) expressing the authentic lysis gene A2 (pQßA2-11). In Fig. 11(A,B), leaking materials were observed through the cell walls. In Fig. 12, these transmission electron micrographs were obtained from the induced cells (K12H1trp) expressing plasmid pQßg-8 in LB medium at 10 (Fig. 12A,B) and 40 (Fig. 12C,D) min after induction. These photographs show that cell profiles were quite homogeneous and noticeably uniform in appearance (Fig. 12B,D). After 10 min (Fig. 12A,B), cell materials inside the cells were denser than those after 40 min (Fig. 12C,D), but leaking material still appeared on the cells in both photographs. Leaking materials observed in the photographs (Fig. 12) could be released from the small holes observed in Fig. 13. In Fig. 12(A), harsh rupture of the host cell was observed, but most cells were not so harshly ruptured (Fig. 12B,D). These cells were grown at lower salt concentration in LB medium, and so most cells did not lyse, but the cell wall structure must be changed to release cytoplasmic materials for an unknown reason. Control cells, which had no lysis gene, showed mostly normal morphology appearance (Fig. 11D).
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H1trp) harbouring plasmid pQßg-8 showed curious cell shapes (Fig. 13). These cells were grown and induced to lyse in LB media containing 1% sodium chloride (Fig. 13A,B,C) or 3% sodium chloride (Fig. 13D–I). They were harvested at 5 min (Fig. 13B) or 20 min (Fig. 13C) or 30 min (Fig. 13D–I) after induction. Figure 13 shows that the contours of the rod-shaped E. coli cells harbouring pQßg-8 remained intact, but were warped morphologically. Also, there were many small holes on the sides of cells (Fig. 13C–E, arrows) and a big breakage (Fig. 13F). These photographs suggest that the cells did lyse upon induction at higher salt concentration in LB medium, but when the cells were grown at lower salt concentration cell wall structures must be changed even if they were not broken with consequent lysis. Figure 13(G,H,I) also shows that there were some connected cells and these connections seemed to be the extruded structures from lysing cells. Arrows show the connected points, and these structures are quite similar to those observed previously for lysis mediated by phage MS2 lysis protein (Nishihara 2003). Figure 13(B) shows two connected cells and similar connections were often observed in later stages. It is not clear whether or not the connected cells, the warped shapes and small holes of the cells observed in the photographs are related. |
Discussion |
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Effective lysis is caused by the product encoded by the frameshift mutation-containing replicase gene. This Rg-lysis protein contains 67 amino acids and is rich in basic amino acids. The strong basic properties of the protein could promote interactions with acidic molecules, particularly nucleic acids, and could have drastic effects. The Rg-lysis protein has short stretches of hydrophobic amino acids in its centre. However, this hydrophobic segment is quite short compared with that of MS2 lysis protein, which is thought to be a membrane protein (Berkhout et al. 1985). It is conceivable that the Rg-protein is a soluble protein rather than being membrane bound. Amino acids 1–24 are the same as that of Qß replicase. Consequently amino acids 25–67, encoded by what in WT (wild-type) is the +1 frame, likely confers the distinctive properties. We compared the sequence of a product predicted by reading the first 67 codons of the WT replicase gene in the +1 frame with the Rg-lysis protein (these 67 codons do not contain a stop codon). The two have similar hydrophobic profiles and isoelectric points (pI:11.70 of the ‘all +1’ and 12.29 of Rg-protein). This similarity means that if an ‘all +1’ product from the start of the replicase gene were to be synthesized, it would likely also have lysis activity. Rg-lysis is, however, dissimilar to the lysis proteins of the group A RNA phages, MS2 and GA, and also to Qß phage A2 protein (data not shown; Mekler 1981; Berkhout et al. 1985; Inokuchi et al. 1986).
With WT RNA phages, cell lysis occurs after a cycle of phage virion maturation and accumulation of phage lysis proteins. However, expression of Rg-lysis protein results in immediate lysis, perhaps because in part of its likely solubility in the cytoplasm. The cell profiles observed in the electron micrographs, and the slow lowering of the growth rate in low salt media of cells harbouring pQßg-8, suggests that most cells only gradually released cell materials. When the cells started to lyse, most of their walls may have been damaged sufficiently that recovery was possible. Alternatively, there may have been holes in the walls which were small enough not to cause drastic cell damage but adequate to permit some cell leakage. However, there is no evidence for either of these explanations. Electron micrographs obtained from the two different types of E. coli cells used (K12H1trp pQßg-8 and JM105 pQßRg-1) were quite different, perhaps because their cell wall structures differ. Qß A2 (maturation/lysis) protein is thought to block cell wall biosynthesis by inhibiting MurA which catalyses murein precursor synthesis, with resulting lysis (Bernhardt et al. 2001b). In contrast, x174 lysis protein E inhibits cell wall synthesis by inhibiting MraY, which catalyses the formation of the first lipid-linked murein precursor (Bernhardt et al. 2000, 2001a). The synthesis of Rg-lysis protein studied here results in very quick cell lysis and its target is likely different than the MruA and MraY targets of the other lysis proteins (Bernhardt et al. 2000, 2001a,b). Future work is needed to ascertain if Rg-lysis acts by attacking cell wall synthesis and, if so, at which step in the pathway.
With sodium chloride incorporated in the media, Rg-lysis synthesis results in immediate cell lysis, especially with high, 4%, sodium chloride. These results reflect the mechanism involved and are different from those of other studies, including ones with GA and SP phages (Nishihara 2002; unpublished results). Cells grown in LB medium without sodium chloride were hypersensitive to EDTA which is known to make the outer membrane unstable by removing magnesium (Voll & Leive 1970). This EDTA effect is similar to the murein-lipoprotein mutant of E. coli described by Suzuki et al. (1978). The EDTA sensitivity observed with Rg-lysis likely reflects damage to the outer membrane.
Does a low level of frameshifting occur naturally on decoding WT Qß replicase to synthesize an equivalent lysis protein?
Many instances of ribosomes shifting the reading frame are erroneous and are a component of the processivity errors in any cell. However, ribosomal frameshifting is utilized for gene expression purposes at some specific sites. In the known cases, signals embedded in mRNA, which are distinct from the shift site, stimulate a high level of frameshifting at the shift site. The frameshifting is said to be programmed and the signals are often known as recoding signals (Gesteland & Atkins 1996). While examples of utilized frameshifting range from HIV to plant viruses and diverse cells from human to E. coli (Namy et al. 2004), quite a number are known in decoding phage genomes. The phages that utilize programmed frameshifting in their expression include T7 (Dunn & Studier 1983; Condron et al. 1991), 
(Levin et al. 1993), P2 (Christie et al. 2002) and a Listeria phage, PSA (Zimmer et al. 2003). Frameshifting also occurs in decoding MS2 to give additional products, including a coat-lysis product (Atkins et al. 1979a; Beremand & Blumenthal 1979; Atkins et al. 2000), but these products are not known to be utilized. The level of certain types of specific frameshifting is known to be influenced by the balance of aminoacylated tRNAs (Gallant & Lindsley 1993) which could change during viral infection. Many of the recoding signals for programmed frameshifting are RNA structures featuring stem loops, pseudoknots, three-way junctions or structures with a triple helical component (Brierley & Pennell 2001). The structure of the RNA of the RNA phages is well known to change during translation and replication. However, not enough is known about the structure of Qß RNA to address whether a suitable recoding signal becomes present later in infection for stimulation of frameshifting in the 5' part of the replicase gene, despite detailed structural analyses (Beekwilder et al. 1995, 1996). For any transframe product encoded in the first part of the replicase gene to have a role in cell lysis, it should be expressed after viral replication. Yet in all single-stranded RNA phages, the replicase start is located in an operator helix to which coat protein dimers bind and inhibit replicase initiation. The structure of this helix has been studied in Qß RNA (Witherell & Uhlenbeck 1989; Spingola et al. 2002). However, the end of the coat readthrough gene is only 22 nt 5' of the replicase start and 10 nt before the SD structure for replicase. Only 5% of ribosomes readthrough the coat gene terminator and with nearly all phages this is only likely to occur at the late stages of infection. It is possible that ribosomes terminating readthrough are, or were (see below), responsible for a low level of initiation at the replicase start site at late stages of replication and that a substantial proportion of these ribosomes shift frame to synthesize a transframe lysis product.
Evolutionary implications
Regardless of whether an equivalent protein to lysis product identified here is ever expressed by wild-type Qß, it has interesting evolutionary implications. Only a short length is required for lysis function, even the C-terminal 32 amino acids of MS2 will suffice (Berkhout et al. 1985). Ribosomal frameshifting to produce a functional Rg-lysis-like protein might have occurred during the early days of Qß phage evolution. It has a similar location as bona fide lysis encoding genes of all the known group A of E. coli RNA phages where the active C-terminal part is encoded by the proximal part of the replicase gene and in the +1 frame with respect to the replicase sequence.
However, Rg-lysis (and presumably any hypothetical equivalent ribosomal frameshifting generated product) lyses cells very quickly, perhaps quicker than optimal for high phage yield. Variants which arose with their lysis gene having some extended structure(s) at the ends or inside the gene, may have been selected. The next step could have been to select for lysis function (by a different mechanism) in the A2 gene. This speculation is consistent with that of Bollback & Huelsenbeck (2001) who proposed that the ancestral RNA phage genome was small and had its lysis gene located around its coat protein and replicase genes. However, it is contrary to the ‘deletion hypothesis’ (Furuse 1987), in which the ancestral RNA phage resembled group B-IV phages, such as SP phage, which have large genomes and their lysis function mediated by their A2 gene (Inokuchi et al. 1988; Priano et al. 1995; Nishihara 2002).
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Experimental procedures |
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Escherichia coli strains K12H1trp
[lacZam, bio-uvrB, (trpE-A)2, rpsL(Nam7, Nam53, cI857, H1)] (Bernard et al. 1979), JM105 (thi, rpsL, endA, sbcB15, hspR4, (lac-proAB), [F',traD36,proAB,lacIqZ M15]) (Yanisch-Perron et al. 1985) and AD202 [F— (argF-lac) U169 araD139 relA1 rpsL150 flbB 5301 deoC1 tsnA 21 thi ptsF 25 ompT::Km] (Akiyama & Ito 1990) were used. As induction of expression from plasmids pGAcl-8 and pSPA2-10 led to quicker lysis with E. coli K12H1trp host cells than E. coli M5219 (Nishihara 2002), K12H1trp cells were mainly used for this analysis. Plasmid pUC18 (Yanisch-Perron et al. 1985) was used for cloning of pQß Rg-1 and plasmid pKK233-2 (Amann & Brosius 1985) was used for expression of pQß Rg-1. Plasmid pPLc245 (Remaut et al. 1983) with slight modifications at the polylinker region with that of pNEB193 (New England Biolabs Inc., MA) was used for cloning and expression of pQßg-8 and pQßA2-11. The polylinker fragment containing restriction enzyme sites AscI and PacI was isolated from pNEB193 with restriction enzymes EcoRI and HindIII, and inserted into pPLc245, which was digested with the same enzymes. The resulting vector was designated as pPLc245 (AP). Plasmid pBAD/Myc-His C (Invitrogen, Carlsbad, CA) was used for addition of a hexa-histidine-tag at the C-terminus of Rg-lysis proteins. Bacteriophage Qß was kindly supplied by T. Watanabe and A. Hirashima (Keio University).
Growth conditions
In all experiments except for growth of E. coli AD202, cells were grown in LB medium (bacto-tryptone, 1%; bacto-yeast extract, 0.5%; sodium chloride, 1%; when not specified) (Sambrook et al. 1989). In some experiments, sodium chloride concentration in LB medium was varied as specified. E. coli AD202 cells were grown in L-broth (polypeptone, 1%; dried yeast extract, 0.5%; sodium chloride, 0.5%). Measuring the optical density at 600 nm monitored cell growth and lysis. E. coli K12H1trp was grown at 28 °C and to induce expression of genes under control of pL of plasmid pPLc245 (AP), the temperature was shifted from 28 °C to 42 °C for thermal inactivation of cI857 repressor molecules. E. coli JM105 was grown at 37 °C and to induce the expression of plasmid pKK233-2, IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to a final concentration of 1 mM. The host strain for plasmid pKK233-2 was E. coli JM105 and that for plasmid pPLc245 (AP) was E. coli K12H1trp. Antibiotics were added to the growth medium when required as follows; streptomycin (25 µg/mL) and ampicillin (50 µg/mL).
Construction of plasmid pQßA2-11 containing Qß lysis/maturation (A2) gene cDNA
RT-PCR amplification of Qß RNA followed the protocols provided with the Promega Access RT-PCR system kit. The forward primer, containing an AscI site and sequence corresponding to position 34–56 of Qß RNA, was 5'-GTAGGCGCGCCAGTACTTCACTGAGTATAAGAGG-3' and the reverse primer, containing a PacI site and the complement of positions 1317–1340 of Qß RNA, was 5'-CGCTTAATTAACAAATTGACCCAAAGTTTCAACGC-3'. The fragments obtained from the RT-PCR amplification were purified with the QIAquick PCR purification kit (Qiagen, Valencia, CA), digested with restriction enzymes AscI and PacI and inserted between the AscI and PacI sites of vector pPLc245 (AP) to give plasmid pQßA2-11.
Isolation of plasmid pQß Rg-1 containing a frameshift mutation in the Qß replicase gene
In 1987, we started to construct a plasmid containing a cDNA copy of the Qß replicase gene. Qß RNA was used as template with reverse transcriptase with the primer, 5'-AAGCTTGGCTGCAGTTTTTTTGGGAGGAGAGAGGGC-3' (complementary to 4202–4217 of Qß RNA) and the product treated with RNase H, E. coli DNA polymerase I and T4 DNA ligase. The resulting ds-DNAs were treated with T4 DNA polymerase to obtain blunt ends and with the restriction enzyme PstI. The resulting DNA fragments were ligated to the HincII and PstI sites of vector pUC18. Derivatives sensitive to the restriction enzymes BstEII and XhoI (which have only targets in replicase gene cDNA), were identified, propagated, and treated with BstEII and PstI. The liberated DNA was ligated with the following synthetic DNA fragments. The synthetic DNA fragments with NcoI and BstEII targets at their ends were ligated with five different kinased oligodeoxyribonucleotides. The resulting fragments were digested with NcoI and BstEII to cleave at the dimerization sites. The resulting NcoI–BstEII fragments consisted of about an 80-nucleotide long duplex. The NcoI–BstEII and BstEII–PstI fragments were ligated to obtain full-length replicase gene cDNA which was cloned into the NcoI and PstI sites in pKK233-2. The resulting plasmid was transformed into E. coli JM105. One of the resulting clones was unusual in that it propagated very slowly. On induction of plasmid expression with IPTG, the cells unexpectedly lysed. The resulting plasmid was designated as pQß Rg-1. The nucleotide sequence of pQß Rg-1 was compared with that of the Qß replicase gene obtained from DDBJ database (accession number X14764). The different positions of the nucleotide sequence at the inserted mutant replicase gene of pQß Rg-1 from that of the replicase gene from the database were as follows with parentheses () indicating the nucleotide number of Qß-RNA from Mekler (1981) and double parentheses (()) indicating numbers from the replicase gene start. G at position ((73)) (2424) was deleted and seven positions were substituted; C to T ((119)) (2471), C to T (2944), G to A (3242), G to A (3289), C to T (3545), G to A (3945) and C to G (4190). The frameshift mutation with the base at position ((73)) deleted, brought a stop codon at nt 202–204 from the start of the replicase gene, into frame.
Construction of plasmid pQßg-8 containing the shorter mutant gene from pQßRg-1 for vector pPLc245 (AP)
PCR amplification of plasmid pQß Rg-1 was with the following primers: The forward primer, containing the AscI site and positions 10–33 from the next of ATG (NcoI site) of pKK233-2, was 5'-GTAGGCGCGCCGAGCGGATAACAATTTCACACAGG-3'. The reverse primer, containing a PacI site and the complement of positions 2599–2622 of Qß RNA, was 5'-CGCTTAATTAACAGCTTCGGTATCAATACCTAGGC-3'. The fragments obtained from the PCR amplification were digested with restriction enzymes AscI and PacI. The resulting fragments were inserted between the AscI and PacI sites of the vector pPLc245 (AP) to give plasmids pQßg-8. The sequences of the constructed plasmids were verified by sequencing (Sanger et al. 1977).
Construction of a fusion plasmid pQM1 containing the Rg-lysis and hexa-histidine-tag encoding sequences
By using the sticky-end PCR method (Zeng 1998), two forward primers 5'-CATGTCTAAGACAGCATCTTCGCG-3', 5'-TCTAAGACAGCATCTTCGCG-3' and a reverse primer 5'-TCCTAAAGTCATCCGGGGTC-3', a fragment containing Rg-lysis encoding sequence was inserted between the NcoI and SnaBI sites of the vector pBAD/Myc-His C (Invitrogen, Carlsbad, CA) to give plasmid pQM1.
Expression and purification of the hexa-histidine-tagged Rg-lysis protein
E. coli AD202 cells were transformed with plasmid pQM1. The transformants were grown in L-broth containing 0.5 M sucrose at 30 °C for 4 h, and then incubated at 30 °C for 24 h with arabinose (0.002%) for the expression of the fusion gene. The hexa-histidine-tagged Rg-lysis protein from 400 mLs of cells was purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (following the Qiagen protocol).
Electron microscopy
For transmission electron microscopy, samples were collected by centrifugation at times indicated in the figure legends and they were washed with PB (phosphate buffer) and fixed overnight at 4 °C by addition of glutaraldehyde to a concentration of 1.5% (w/v). After washing with PB, samples were suspended in PB and embedded in 4% of agarose. After post-fixation in 1% (w/v) osmium tetroxide for 60 min at room temperature, the cells were washed with PB and dehydrated with alcohol. Cells were then infiltrated and embedded in Spurr low-viscosity resin (Spurr 1969) and polymerized, cut with a diamond knife on a Reichert-Nissei Ultracut Microtome, and mounted on copper grids coated with Formvar films and stained with uranyl acetate and lead citrate. Ultrathin sections were examined with a JEM-2000EXII (Jeol Ltd, Japan) transmission electron microscope.
For scanning electron microscopy, samples (0.5 mL) were taken at the indicated times in the figure legend for electron microscopic analysis. They were washed with PB and fixed overnight at 4 °C by addition of glutaraldehyde to a concentration of 1.5% (w/v). After washing with PB, samples were suspended in PB and streaked on plastic sheets coated with calf dermis collagen (Celltight Celldesk; Sumilon, Japan) for 10 min. After post-fixation in 1% (w/v) osmium tetroxide for 30 min at room temperature, the cells were dehydrated with alcohol. Dried samples were coated with platinum and they were examined with a field emission scanning electron microscope JSM-6340F (Jeol Ltd, Tokyo, Japan).
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Acknowledgements |
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H1trp and plasmid pPLc245 were kindly provided by LMBP (University of Gent, Belgium) and E. coli JM105 was kindly provided by T. Takeshita (Ajinomoto Co. Inc.). We also thank S. Fujisaki and S. Sugimoto for helpful suggestions in performing experiments, and H. Hayashi, T. Kishimoto and Y. Makino for the mass spectrometric analysis. JFA was supported by NIH grant GM48152 and an award from the Science Foundation Ireland. |
Footnotes |
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Present addresses:aShowa2-chome, 4-23-1101, Kurashiki City, Okayama, 710-0057, Japan;
bAminoscience Laboratories, Global Foods & Amino Acids Company, Ajinomoto Co., Inc. 1-1, Suzuki-cho, Kawasaki City, 210-8681, Japan
* Correspondence: E-mail: tnishihara{at}ma.0038.net |
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Received: 29 June 2004
Accepted: 15 July 2004
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