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1 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
2 Division of Insect Sciences, National Institute of Agrobiological Sciences, Owashi 1-2, Tsukuba, Ibaraki 305-8634, Japan
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Introduction |
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The red blood mutant is a body color mutant of B. mori, which is regulated by a single recessive gene called rb (Inagami & Akagi 1954; Makino et al. 1954). Grown larvae of the rb strain display reddish skin (Fig. 1B), in contrast to the normal white body (Fig. 1A). Hemolymph of rb mutant larvae becomes red when exposed to air, whereas hemolymphs of normal strains become black during melanization (Fig. 1C). Although rb was mapped on to the proximal end of the 21st linkage group of B. mori by Doira et al. (1977), the candidate gene itself has not been identified.
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Results |
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The body color mutant of the silkworm, rb, was discovered over 50 years ago (Makino et al. 1954). The hemolymph of this mutant becomes red when exposed to air. Makino et al. (1954) reported that the irregular color of this mutant was due to abnormal accumulation of 3-HK. Thus, we speculated that the rb strain might lack the hydrolysis activity or pathway from 3-HK to 3-HAA. Although this reaction is catalyzed by the enzyme KYNU in microorganisms and animals, it has not yet been discovered in insects. To identify the gene responsible for the rb phenotype, we first examined whether KYNU activity was observed in tissues of the fifth instar larvae. Proteins extracted from the Malpighian tubules, fat body, and hemolymph of day 4 fifth instar larvae were used in the analysis. In Malpighian tubules, protein extracts from normal strains (+/+) showed high levels of KYNU activity to the substrate of L-KYN (Fig. 1D). KYNU activity in the heterozygous individuals (rb/+) was approximately 70% of that in normal homozygotes (+/+). These results strongly suggest that Bombyx has the hydrolysis pathway from 3-HK to 3-HAA and the KYNU may be encoded by the Bombyx genome. In addition, we observed that the KYNU activity was significantly reduced in the rb strain (rb/rb) with levels as low as 5% in normal individuals (+/+), suggesting the fact that abnormal accumulation of 3-HK in rb strain likely results from the loss of KYNU activity.
Cloning of KYNU gene from Bombyx mori
We performed homology searches against B. mori EST data bases and found an EST clone maV32451 from the cDNA library prepared from the Malpighian tubules of fifth instar larvae (p50T) (Mita et al. 2003). We sequenced the clone and obtained a full length cDNA that showed significant homology to bacterial Kynu genes. This gene, BmKynu, consisted of an open reading frame (ORF) of 1278 bp, which encodes a 426-aa protein with a predicted molecular mass of 48.6 kDa (Fig. 2). Whole-genome shotgun data revealed that BmKynu cDNA was completely covered in Contig BAAB01012814 (Mita et al. 2004) and Contig AADK01006945 (Xia et al. 2004), and this gene did not have any introns. Using the Signal P program (http://www.cbs.dtu.dk/services/SignalP/), it was predicted that the BmKYNU polypeptide or the KYNUs of other organisms would not contain any hydrophobic signal peptide.
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Next, we cloned the BmKynu genes of three rb mutant strains and seven normal strains using genomic DNAs and cDNAs. We compared their nucleotide sequences in the coding region and deduced the amino acids (Fig. 2). We found that nucleotide sequences of five normal strains, e36, l70, a65, i41 and B, were identical with that of p50T, whereas 16 nucleotide substitutions, three of which resulted in amino acid substitution at residues 54, 154 and 315, were observed in the normal strain Sho-on. However, only a single nucleotide mutation (C305T) was found in the BmKynu ORF of each rb strain (k20, No. 771 and No. 772), which substituted isoleucine for threonine at residue 102. These results indicated that mutations at residues 54, 154, and 315 in BmKYNU were not associated with the rb phenotype. Previous studies showed that the cofactor PLP binding site, Lys-237, and four more residues contributing to PLP binding, Asp-133, Asp-211, Tyr-236 and Trp-265, were commonly conserved in both bacterial and human KYNUs (Momany et al. 2004) (Fig. 3A). An amino acid residue, Thr-102, which is mutated to Ile in BmKYNU of rb strain, was not well-conserved in other organisms.
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BmKYNU exhibited high homology to bacterial KYNUs with 54% amino acid identity to Alkaliphilus oremlandii KYNU (GENBANK accession number ABW20264 [GenBank] ), whereas only 33% residues were identical to human KYNU isoform b (Toma et al. 1997; GENBANK accession number NP_001028170) (Fig. 3A). To investigate the evolutionary relationship between BmKYNU and KYNUs in other organisms, phylogenetic analysis was performed. When the BLAST search was conducted in the public protein data bases, many bacterial, fungal, and animal KYNUs were retrieved. A neighbor-joining tree showed that BmKYNU was clustered into the bacterial lineages and separated from fungal or animal ones (Fig. 3B). We also performed a BLAST search using several genome/EST data bases of other insects but did not find any BmKYNU homologues in these insects (Table 2). These results suggest that an ancestral insect might have lost the Kynu gene by evolution; however, B. mori had presumably acquired BmKynu by a horizontal gene transfer from bacteria.
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We investigated the spatiotemporal expression pattern of BmKynu mRNA by RT-PCR (Fig. 4). Total RNA was isolated from tissues of the fourth molting (Fig. 4A) and day 3 of fifth instar (Fig. 4B), as well as from the Malpighian tubules from day 2 of fourth instar to wandering stages (Fig. 4C). We found that BmKynu was mainly expressed in the larval Malpighian tubules and was consistent with a strong KYNU activity in this tissue (Fig. 1D). No significant differences were observed in mRNA expression levels between normal and rb strains, suggesting that the KYNU activities, but not KYNU expression levels, might differ in larval tissues of normal and rb strains.
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To compare the enzymatic activity of normal and mutant BmKYNUs, we expressed three His-tagged recombinant proteins using a bacterial expression system, including two wild-type BmKYNUs from p50T and Sho-on and a mutant type from the rb strain. The three recombinant proteins were purified using nickel chromatography columns and confirmed by an immunoblot analysis using an anti-His antibody. The proteins had identical molecular masses of approximately 49 kDa, a result consistent with the predicted molecular mass (data not shown).
As expected, two wild-type proteins exhibited high enzymatic activity to the substrates of both L-KYN and 3-hydroxy-DL-KYN (Fig. 5). No significant difference in the KYNU activity was found between the two wild-type proteins; however, the activity of the mutant type was less than 20% when compared to that of wild-type BmKYNUs. These data strongly indicated that the single amino acid mutation, T102I in BmKYNU, significantly reduced its activity. In vivo and in vitro enzymatic assays clearly demonstrated that a decreased BmKYNU activity was presumably correlated with the morphological red coloration in the rb mutant strain (Figs 1D, 5).
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To determine the consistency between the rb phenotype and the BmKynu genotype, we performed linkage analysis between p50T (+/+) and No. 771 (rb/rb) using a routine crossing method (Table 3). We obtained F1 progenies between p50T (+/+) and No. 771 (rb/rb), and backcrossed the F1 hybrid to females of No. 771. The phenotypes of 188 larvae obtained from the backcrossed individuals were confirmed by both body color and hemolymph coloration pattern when exposed to air for 10 min (see Fig. 1). Genomic DNA was isolated and analyzed by genomic-PCR using BmKynu-specific primers (Table 1). We sequenced all the PCR products and detected a single SNP at nt 305 in BmKynu ORF. Corresponding to nt 305C in normal BmKynu (+/+) (Fig. 6A), nt 305 in 94 BC1 individuals of rb/+ was heterozygous T/C (Fig. 6B) and homozygous T in 94 rb/rb individuals (Fig. 6C). The results showed that no recombination between rb and BmKynu was detected among 188 progenies, suggesting that the candidate gene BmKynu corresponds to rb.
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Discussion |
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BmKynu is the first KYNU gene in insects. Interestingly, BmKYNU exhibited high homology to bacterial KYNUs (Fig. 3A). Analysis of the genomic and cDNA sequences of BmKynu showed that there was no intron in its coding region. The gene structure of BmKynu is very similar to bacterial Kynus; in human Kynu, however, there are 11 introns in isoform a and 8 in isoform b (Alberati-Giani et al. 1996; Toma et al. 1997). By phylogenetic analysis, BmKYNU was shown to belong to bacterial lineages and far separated from KYNUs of the metazoans, including cnidarians and nematodes (Fig. 3B). Furthermore, we could not obtain any KYNU from genome/EST data bases of other insects (Table 3). These results may suggest that an ancestral arthropod or insect had lost the gene during the evolutionary process, but B. mori reacquired the gene through a horizontal transfer from bacteria. Although homologous genes were not observed in EST data bases of several other lepidopteran insects, we cannot conclude at present whether the gene is specific in Bombyx as the EST data bases for other lepidopteran insects are not large enough to find most of the expressed genes in each insect (Table 3).
BmKynu was mainly expressed in the larval Malpighian tubules (Fig. 4), where we observed high KYNU activity in larvae of normal strains (Fig. 1D). Cloning and DNA sequencing of BmKynus from 7 normal and 3 rb strains revealed that BmKynu has been maintained as an allelic heterogeneity in different B. mori strains (Fig. 2). Three amino acid substitutions did not influence the KYNU activity of p50T and Sho-on, however, the single amino acid mutation of T102I found in BmKYNU of rb strains significantly reduced both the in vivo and in vitro activities (Figs 1D, 5). Corresponding residues to Thr-102 in other organisms are poorly conserved, e.g. Thr-97 in Pseudomonas fluorescens and Leu-137 in Homo sapiens (Fig. 3A). However, three-dimensional structure showed that Thr-97 in P. fluorescens is one of the residues surrounding the cofactor PLP moiety, and potentially donates hydrogen bond to a phosphate oxygen of PLP, as well as Leu-137 in H. sapiens (Momany et al. 2004; Lima et al. 2007). Moreover, Thr-97 in P. fluorescens lies at the onset of helix H6, which is just near the combining position of two monomers to form a dimmer (Momany et al. 2004). These studies suggest that Thr-102 might similarly involve in stabilizing the PLP cofactor and proper folding of BmKYNU. Unexpectedly, the residue corresponding to Thr-102 in A. oremlandii KYNU is Ile (Fig. 3A). Although we could not provide a mechanistic explanation that this substitution results in reduction of KYNU activity in B. mori, our present data suggest that Thr-102 is an important residue in BmKYNU function. Deficiency of KYNU will accelerate the accumulation of a large amount of 3-HK in vivo (Makino et al. 1954; Ishiguro et al. 1971). Our data clearly showed that the reduction in BmKYNU activity is responsible for the red larval skin in the rb strains. A similar case in humans is a genetic disease known as xanthurenic aciduria. A substitution of Thr-198 with Ala in human KYNU causes a deficiency in KYNU activity, resulting in excessive urinary excretion of xanthurenic acid (transaminated product of 3-HK), 3-HK, and KYN (Christensen et al. 2007).
KYN is one of the endogenous metabolites of tryptophan. In mammals, there are three downstream pathways for KYN metabolism, two of which are the hydrolysis pathway whereby KYNU produces AA and the transamination pathway whereby KYN aminotransferase (KAT) is converted to kynurenic acid. The major reaction is the production of 3-HK by the catalysis of KYN 3-monooxygenase (KMO). 3-HK can also be catalyzed by KYNU and KAT to produce 3-HAA and xanthurenic acid, respectively (Bertazzo et al. 2001; Stone & Darlington 2002) (see Fig. 7). Insects have been supposed to lack both the glutamate and nicotinic acid pathways (Linzen 1974; Kayser 1985). NAD is well-known as an important coenzyme in a variety of metabolic reactions. NAD biosynthesis pathway from 3-HK requires at least three enzymes in this order: KYNU, 3-hydroxyanthranilic acid 3,4-dioxygenase (HADO), and quinolinic-acid phosphoribosyl transferase (QPRT) (Fig. 7). In the present study, we discovered a KYNU gene from the silkworm, B. mori, suggesting that B. mori had uniquely acquired the KYNU-associated reactions that are present in mammals and microorganisms but not in most other insect species. However, we did not find HADO- and QPRT-coding genes in the genome of B. mori, Drosophila melanogaster, or Anopheles gambiae (Zdobnov et al. 2002; Mita et al. 2004; Xia et al. 2004). The results suggest that B. mori, like other insects, is probably unable to synthesize NAD through the de novo pathway from endogenous tryptophan. Instead, insects might synthesize NAD through an alternate pathway by utilizing exogenous niacin (nicotinic acid and nicotinamide) from their diets, as reported in some yeast species (Li & Bao 2007). Therefore, we think that a possible downstream pathway of 3-HAA metabolism in B. mori is to form cinnabarinic acid by autoxidation or by catalysis using some catalases (Christen et al. 1992; KEGG, http://www.genome.jp/kegg/pathway.html), a claim which is supported by the results of a previous study performed on B. mori Malpighian tubules (Ogawa et al. 1983) (Fig. 7).
Although tryptophan is an essential amino acid for insects, it is harmful in excessive levels (Kayser 1979). Cerstiaens et al. (2003) also reported that tryptophan and its metabolites were toxic to primary cultures of insect neurons; injection into adult flies caused severe motor dysfunction. As 3-HK is easily oxidized under physiological conditions, it can induce apoptosis of neural cells at micromolar concentrations and can stimulate formation of reactive oxygen species (Wei et al. 2000). Therefore, it is important for insects to balance the levels of tryptophan and its metabolites in their bodies. In mosquitoes, the hydrolysis of 3-HK does not occur due to the lack of KYNU. However, mosquitoes prevent the accumulation of 3-HK by converting the chemically reactive 3-HK to the chemically stable xanthurenic acid via transamination, probably causing the detoxification of 3-HK (Li et al. 1999; Han et al. 2002, 2007; Rossi et al. 2005, 2006). In view of this concept, we speculate that a physiological role of BmKYNU is to detoxify tryptophan metabolites, although any abnormal phenotypes other than the red body coloration have not been observed in rb strains. Moreover, we found that a KAT homologue of B. mori was highly expressed in the EST libraries (Mita et al. 2003), suggesting that transamination of KYN/3-HK to kynurenic acid/xanthurenic acid might also be important in B. mori to control the levels of KYN and 3-HK, especially in rb silkworms.
The larval body color in B. mori is determined by concentration of melanin in the cuticle and xanthommatin, sepialumazine, sepiapterin, and uric acid in the epidermis (Mazda et al. 1980; Ohashi et al. 1983; Komoto 2002; Kato et al. 2006). Inagami (1954) reported that the red pigment in the rb strain was not hallachrome, a red substance derived from tyrosine, but melanin. Therefore, he assumed that the red coloring material was produced from both metabolites of tryptophan and tyrosine. On the other hand, Maki et al. (1995) concluded that the red pigment detected in the integument of feeding larvae and hemolymph of spinning larvae was xanthommatin because the red pigment extracted from rb larvae showed identical absorption spectrum to that of synthesized xanthommatin. 3-HK highly accumulated in the rb strain because of the loss of KYNU activity is a direct precursor of xanthommatin. Thus, it is possible that a major component of the red pigment is xanthommatin (ommochrome), which changes the color of skin and the melanization pattern of hemolymph (Fig. 1B,C).
Among the many types of endogenous pigments in insects, melanin synthesized from tyrosine, ommochromes from tryptophan, and pteridines from guanosine triphosphate are the main contributors to body coloration (Kato et al. 2006). The identity of most of the molecules participating in these pigmentation pathways is still unclear. Ommochromes are not only major eye pigments for insects (Linzen 1974; Li et al. 1999), but in Lepidoptera, they are also widely used in the formation of larval and adult body color and marking, which are important ecologic characters regulated by many molecules (Nijhout 1991; Reed & Nagy 2005; Kato et al. 2006). This study adds a new regulator, BmKynu, to the pigmentation network in B. mori. Normal expression of BmKynu can prevent the silkworm from showing a red larval body color. In addition to rb, there are many B. mori mutants of varying body color and marking, such as ch, so, q, Sel, lem, and Ze (Banno et al. 2005; Silkworm Base, http://www.shigen.nig.ac.jp/silkwormbase/index.jsp). With the release of newly integrated B. mori genome sequence data (KAIKObase, http://sgp.dna.affrc.go.jp/KAIKObase/), these mutants can be well-examined in a few years, a scientific advancement that will greatly contribute to our understanding of the molecular mechanisms of insect color pattern formation.
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Experimental procedures |
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Three rb strains were used in this study; k20 was obtained from Kyushu University, and No. 771 and No. 772 from National Institute of Agrobiological Sciences. Seven normal strains were used in this study; Four of them, e36, l70, a65, and i41, were also obtained from Kyushu University and the remaining three, p50T, Sho-on, and B, are maintained in our laboratory. All larvae were fed with fresh mulberry leaves under normal conditions (12 L:12D, 25 °C).
Protein extract from tissues
The Malpighian tubules, fat body, and hemolymph were collected from five individuals of the fifth instar larvae on day 4. The tissue was homogenized in 10 mM PBS (pH 7.0) containing a mixture of proteinase inhibitors (Roche). The supernatant of homogenates was collected by centrifugation (10 000 g, 4 °C, 10 min) and subjected to the PD-10 column (Amersham Biosciences) for desalting and exchanging the buffer with Tris-HCl (pH 8.0, 10 mM). The resultant solution was concentrated using Amicon Ultra centrifugal filter devices (Millipore). Protein concentration was estimated using a Coomassie Plus Protein Assay Reagent Kit (Pierce Biotechnology) with bovine serum albumin used as a standard.
Cloning of Bombyx mori kynureninase gene
To identify the Bombyx gene showing homology to kynureninase (Kynu) gene, we screened EST data bases (Mita et al. 2003) and genome sequences (Mita et al. 2004; Xia et al. 2004) using the BLAST program. By sequencing cDNA and genomic clones, we obtained the sequence of the full-length BmKynu that encodes a putative KYNU. The nucleotide sequences reported in this article have been submitted to the GenBankTM./EBI Data Bank with accession numbers AB441721 [GenBank] (BmKynu-p50T), AB441722 [GenBank] (BmKynu-Sho-on), and AB441723 [GenBank] (BmKynu-rb).
Total RNA isolation and RT-PCR analysis
Total RNA was extracted from tissues of normal and rb strains as previously described (Meng et al. 2008). The first-strand cDNA was synthesized from 1 µg of the total RNA using a TaKaRa RNA PCR Kit (TaKaRa). Expression profiles of BmKynu in different stages or tissues were analyzed by reverse-transcription PCR (RT-PCR). The PCR condition was 94 °C for 2 min followed by 30 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 1 min. The PCR primers used in the experiment are listed in Table 1. PCR products were electrophoresed on 1.0% agarose gels.
Phylogenetic analysis
Amino acid sequences were aligned using the CLUSTALX program (Thompson et al. 1997). A neighbor-joining tree was constructed using the NEIGHBOR program in PHYLIP (Felsenstein 1989). The reliability of the tree was tested by bootstrap analysis with 1000 replications.
Expression and purification of recombinant BmKYNU
To construct bacterial expression vectors, the coding regions of wild-type A (wtA, p50T type), wild-type B (wtB, Sho-on type), and mutant type (mut, rb type) BmKynus with a 6xHis-tag sequence at the C-terminus, were amplified by PCR from the corresponding cDNA templates. The primers used are listed in Table 1. PCR products were digested with HindIII and ligated into the pET24b vector (Novagen), resulting in three recombinant expression vectors, pET/BmKYNU_wtA, pET/BmKYNU_wtB, and pET/BmKYNU_mut. They were transformed into Escherichia coli strain BL21(DE3) competent cells and grown at 37 °C in 100 mL of Luria–Bertani medium containing 20 µg/mL kanamycin. BmKYNU expression was induced by the addition of isopropyl-1-thio-
-D-galactopyranoside (IPTG) to a final concentration of 1 mM. The transformants were cultured at 15 °C overnight after IPTG induction. The cells were collected by centrifugation (2000 g, 4 °C, 10 min), and suspended in 10 mL of B-PER bacterial protein extraction reagent (Pierce) containing a cocktail of proteinase inhibitors (Roche). The supernatant was collected by centrifugation (6000 g, 4 °C, 15 min) and His-tagged BmKYNUs were purified using the His GraviTrap nickel affinity column (GE Healthcare) according to the manufacturer's instructions. After the column was equilibrated with 10 mL of binding buffer (20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, pH 7.4), a protein sample was loaded on to the column and washed with 10 mL of the binding buffer. Elution was performed using an elution buffer (20 mM sodium phosphate, 500 mM NaCl, 200 mM imidazole, pH 7.4). Finally, the eluate was desalted and concentrated. Protein concentration was then determined as described above.
Immunoblot analysis
Expression of recombinant BmKYNU was analyzed by immunoblot analysis using anti-His antibody (Qiagen) as previously described (Daimon et al. 2008). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Laemmli 1970), the proteins were electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon-P, Millipore) using a blotting apparatus (Atto) at 2 mA/cm2 for 1 h in transfer buffer (100 mM Tris, 192 mM glycine, 20% methanol). The blot was blocked in a TBS-T buffer (20 mM Tris–HCl, pH 7.6, 137 mM NaCl, 0.1% Tween-20) containing BlockAce (DS Pharma Biomedical Co., Ltd) for 30 min followed by 1 h incubation in TBS-T containing anti-His antibody (1 : 5000 dilution). The membrane was then incubated in TBS-T containing the secondary antibody, goat anti-mouse IgG-HRP conjugate (Zymed) 1 : 5000 dilution, for 1 h and visualized using Western Lightning Chemiluminescence Reagent Plus (PerkinElmer) and a LAS 1000 imaging system (Fuji Film).
Enzyme assay
KYNU activity was assayed according to the method reported by Tanizawa et al. (1976) with some modifications. Ten micrograms of proteins from tissue or 0.5 µg of recombinant BmKYNU was used in the assessment. The standard reaction mixture consisted of 10 µM PLP, 25 µM L-KYN, or 3-hydroxy-DL-KYN (Sigma), 2.5 mM Tris–HCl (pH 8.0), and the enzyme in a final volume of 100 µL. The reaction was initiated by the addition of the enzyme and kept at 37 °C for 10 min. KYNU activity was determined by measuring the rate of decrease in absorbance at 360 nm for L-KYN or 370 nm for 3-hydroxy-DL-KYN because of their hydrolysis. Reaction without addition of the enzyme was used as a control. One unit of enzyme was defined as the amount of the enzyme per microgram of protein that catalyzed the hydrolysis of 1 nmol of substrate per minute (nmol/min/µg). The data was analyzed by one-way ANOVA followed by Dunnett's test to localize the significant difference. A P-value of < 0.05 was considered significant and a value < 0.01 was considered extremely significant.
Linkage analysis
To determine the recombination value between rb and BmKynu, we performed genetic linkage analysis by a routine method. Normal strain p50T and rb strain No. 771 were used. The F1 male moths of p50T and number 771 were backcrossed to number 771 females. A total of 188 BC1 individuals (rb/rb 94, +/rb 94) were used in the linkage analysis. Phenotypic characters of each individual were identified by both body color and hemolymph coloration in air. Genomic DNA was isolated using the Wizard SV 96 genomic DNA purification system (Promega) as described in the recommended protocol. Single nucleotide polymorphism observed in BmKynu at nt 305 (see Figs 2, 6) was investigated by genomic-PCR and direct sequencing of the PCR products. Primers used are listed in Table 1. DNA sequences were determined using an ABI Big Dye Terminator Cycle Sequencing Ready Reaction Kit version 3.1 (Applied Biosystems) and an ABI Prism 3130 Genetic Analyzer (Applied Biosystems).
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Acknowledgements |
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* Correspondence: shimada{at}ss.ab.a.u-tokyo.ac.jp
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Received: 29 September 2008
Accepted: 23 October 2008
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