Intraspecific polymorphism in peroxidase genes located on Arabidopsis thaliana chromosome 5

Nucleotide sequences of five peroxidase genes AtPrx52-AtPrx56 located on the upper arm of chromosome 5 were comparatively analyzed for six Arabidopsis thaliana ecotypes and lines (Columbia, Dijon-M, Blanes-M, Enkheim-M, Ler, and K-156). The level of intraspecific nucleotide variation significantly differed between these genes (up to 20 times): the greatest diversity was observed in the tandemly duplicated AtPrx53 and AtPrx54 genes, while the least variation was observed in AtPrx56 gene. AtPrx53 and AtPrx54 genes demonstrated allelic dimorphism and nonrandom association of polymorphic sites was demonstrated for AtPrx54 gene. A relationship has been revealed between haplotypes of these genes and the mobility of anionic peroxidase isoforms. Since the haplotypes of AtPrx53 gene code for proteins with two nonconservative amino acid substitutions, different mobility of anionic peroxidase isoforms could be due to diallelic polymorphism in the amino acid sequence of AtPrx53 protein.     

Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.     

Tracing the history of an enzyme polymorphism: the case of alcohol dehydrogenase-2 (Adh-2) of the olive fruit fly Bactrocera oleae

In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.     

Analysis of expression profiles of three peroxidase genes associated with lignification in Arabidopsis thaliana

We have investigated the mechanism of lignification during tracheary element (TE) differentiation using a Zinnia elegans xylogenic culture. In the process, we isolated ZPO-C , a peroxidase gene of Z. elegans that is expressed specifically in differentiating TEs. ZPO-C is suggested to be involved in lignification of Z. elegans TEs in vivo and in vitro. Furthermore, a peroxidase gene of Arabidopsis thaliana ( AtPrx66 ), which is homologous to ZPO-C , was identified. The expression profile and functions of the gene in planta remain to be investigated. In this study, we performed promoter :: β-glucuronidase (GUS) assays to investigate the expression profiles and functions of the ZPO-C -like peroxidases in A. thaliana . We generated transgenic A. thaliana lines carrying AtPrx66, AtPrx47 or AtPrx64 (peroxidases showing high sequence similarity to AtPrx66 ) promoter :: GUS reporter gene fusions. The GUS activities of AtPrx66, AtPrx47 and AtPrx64 promoter :: GUS lines were arranged concentrically from the center to the periphery in the roots of seedlings. Furthermore, histochemical GUS assays using inflorescence stems showed that AtPrx66, AtPrx47 and AtPrx64 promoter-driven GUS were mainly expressed in the differentiating vessels, xylem parenchyma and sclerenchyma, respectively. These results suggest that the gene expressions of these three peroxidases, which showed high sequence similarity to one another, are differentially regulated in various tissues and organs. In addition, our results suggest that while AtPrx66 and AtPrx47 are associated with lignification of vessels, AtPrx64 is associated with lignification of sclerenchyma.     

Genetic polymorphisms and haplotype structures of the CYP4A22 gene in a Japanese population

The CYP4A fatty acid monooxygenases oxidize endogenous arachidonic acid to 20-hydroxyeicosatetraenoic acid that acts as a regulator of blood pressure. Among the isoforms of the CYP4A subfamily, the human CYP4A22 was recently identified. In this study, we report the comprehensive investigation of polymorphisms in the CYP4A22 gene. To investigate genetic variation in CYP4A22 in 191 Japanese subjects, we used denaturing HPLC (DHPLC) and direct sequencing. Our investigation has enabled the identification of 13 sequence variations in the CYP4A22 coding region, thereby demonstrating for the first time that this gene is subject to polymorphism. Two of these sequence variations correspond to silent mutations located in exons 8 (His323His) and 9 (Gly390Gly). Nine of these sequence variations correspond to missense mutations located in exons 1 (Arg11Cys), 3 (Arg126Trp), 4 (Gly130Ser and Asn152Tyr), 5 (Val185Phe), 6 (Cys231Arg), 7 (Lys276Thr), 10 (Leu428Pro), and 12 (Leu509Phe). One of these sequence variations corresponds to nonsense mutations located in exon 9 (Gln368stop). The 13th mutation corresponds to a nucleotide deletion (G7067del) that causes a frameshift and consequently results in a stop codon 80 nucleotides downstream. In addition to the wild-type CYP4A22*1 allele, 20 variants, namely CYP4A22*2-15, were characterized by haplotype analysis. Based on these data, we concluded that allelic variants of the human CYP4A22 gene exist and speculated that some of these variants may be functionally relevant.     

Exon variability of gene encoding glycerol-3-phosphate dehydrogenase of Ixodes ricinus ticks

We have previously found apparent differences in Gpdh allele frequences between borrelia infected and uninfected Ixodes ricinus as revealed by native gel electrophoresis of allozyme polymorphisms. The present study deals with the genetic basis of the observed allozyme polymorphism. Multiple sequence alignment of 36 Gpdh open reading frames identified a total of 40 polymorphic nucleotide sites. Of the 40 polymorphic nucleotide sites, 34 were silent (did not result in amino acid residue change), while six were active causing a change in the amino acid chain. All polymorphic amino acid sites were situated within the N-terminal NAD-binding domain, whereas the C-terminal substrate-binding domain was highly conserved. Analysis of the obtained Gpdh sequences and GPDH allozyme polymorphisms for individual ticks pointed to amino acid changes at positions 61 (glycine-to-glutamic acid), 64 (serine-to-cysteine) and 102 (glycine-to-arginine) as a key for differential mobility of GPDH allozymes in an electric field. Our findings are discussed in the context of the molecular basis of I. ricinus host finding behavior.     

Purification and cloning of a Chinese red radish peroxidase that metabolise pelargonidin and forms a gene family in Brassicaceae

An anionic peroxidase RsPrx1 was purified (RZ=3.0) and characterized from roots of Chinese red radish (Raphanus sativus var. niger, Brassicaceae). The specific activity of RsPrx1 (micromol mg(-1) min(-1)) is 413.5 (ferulic acid); 258.7 (ABTS); 177.3 (caffeic acid) and 10.0 (guaiacol acid). The optimum pH is 4.0 (citrate buffer) using ABTS as substrate. RsPrx1 can utilise the red pigment present in the root, pelargonidin, as substrate and the specific activity is 93.6 micromol mg(-1) min(-1). The molecular mass of RsPrx1 is 45 kDa (denatured) and 46 kDa (native) as determined by SDS-PAGE and gel filtration, respectively. The isoelectric point (pI) determined by native IEF is 4.7 and by chromatofocusing (Mono P) is 5.1. Analysis of tryptic peptides by nanoscale liquid chromatography-tandem mass spectrometry (LC-MS/MS) covered 27% of the RsPrx1 sequence and confirmed its identity. The gene encoding RsPrx1 was cloned by PCR and the amino acid sequence showed the highest identity (82%) to peroxidase AtPrx22 and AtPrx23 from Arabidopsis thaliana and to HRPC3 and HRPE5 from horseradish, respectively. Activity-stained IEF gels show that RsPrx1 is primarily expressed in the roots in agreement with the expression profile of the orthologous genes. These five orthologous peroxidases have three introns of variable length and sequence at conserved locations between the distal and proximal histidine. The results suggest that RsPrx1 orthologs are widespread in the Brassicaceae plant family with a 15-residue-long C-terminal propeptide in common. Based on the results, we propose that RsPrx1 and orthologs are targeted to the vacuoles to modify stored anthocyanins like pelargonidin.     

Purification and identification of a Ca(2+)-pectate binding peroxidase from Arabidopsis leaves

A protein fraction was obtained from Arabidopsis (Arabidopsis thaliana, L.) leaf extract by affinity chromatography through a Ca(2+)-pectate/polyacrylamide gel. Further purification by preparative isoelectric focusing and SDS PAGE allowed the separation of a peroxidase that was identified as being peroxidase AtPrx34 (AtprxCb, accession number X71794) by N-terminal amino acid microsequencing. AtPrx34 belongs to a group of five Arabidopsis sequences encoding putative pectin-binding peroxidases. An expression study showed that it is expressed in root, stem, flower and leaf. It was produced by Escherichia coli and tested for its ability to bind to Ca(2+)-pectate. The identity of the amino acids involved in the interaction between the peroxidase and the Ca(2+)-pectate structure is discussed.     

Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium.

Two amylases have been purified from the culture fluid of an alkaliphilic bacterium. Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage. Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose. A-60 and A-180 are immunologically unrelated enzymes. The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined. An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein. The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria. Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases. The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases. It appears, therefore, that A-180 has arisen by gene fusion events.     

Identification of a residue responsible for UDP‐sugar donor selectivity of a dihydroxybenzoic acid glycosyltransferase from Arabidopsis natural accessions

UDP‐glycosyltransferase (UGT) plays a major role in the diversity and reactivity of plant specialized metabolites by catalyzing the transfer of the sugar moiety from activated UDP‐sugars to various acceptors. Arabidopsis UGT89A2 was previously identified from a genome‐wide association study as a key factor that affects the differential accumulation of dihydroxybenzoic acid (DHBA) glycosides in distinct Arabidopsis natural accessions, including Col‐0 and C24. The in vitro enzyme assays indicate that these distinct metabolic phenotypes reflect the divergence of UGT89A2 enzyme properties in the Col‐0 and C24 accessions. UGT89A2 from Col‐0 is highly selective toward UDP‐xylose as the sugar donor, and the isoform from C24 can utilize both UDP‐glucose and UDP‐xylose but with a higher affinity to the glucose donor. The sequences of the two isozymes only differ at six amino acid residues. Examination of these amino acid residues in more natural accessions revealed a strong correlation between the amino acid polymorphism at position 153 and the DHBA glycoside accumulation pattern. Site‐directed mutagenesis that swapped residue 153 between UGT89A2 from Col‐0 and C24 reversed the UDP‐sugar preferences, indicating that residue 153 plays an important role in determining sugar donor specificity of UGT89A2. This study provides insight into the key amino acid changes that confer sugar donor selectivity on UGTs, and demonstrates the usefulness of natural variation in understanding the structure–function relationship of enzymes involved in specialized metabolism.     

Molecular cloning and characterization of a chromosomal gene for human eosinophil peroxidase

Using human myeloperoxidase cDNA as a probe, a chromosomal gene related to myeloperoxidase was isolated from a human gene library. Comparison of the amino acid sequence deduced from the nucleotide sequence of the cloned gene with that of human eosinophil peroxidase purified from buffy coats has indicated that the isolated gene is the chromosomal gene for human eosinophil peroxidase. Like human myeloperoxidase gene, human eosinophil peroxidase gene consists of 12 exons and 11 introns spanning about 12 kilobases. The gene can code for a protein of 715 amino acids with a calculated Mr of 81,036. The heavy chain and the light chain of eosinophil peroxidase were located on the COOH and NH2 terminus of the protein, respectively. The coding sequences of eosinophil peroxidase and myeloperoxidase show homologies of 72.4% at the nucleotide and 69.8% at the amino acid level, while little homology was found in the 5'-flanking region. Northern hybridization and S1 mapping analysis of RNA from human leukemic cells have indicated that the eosinophil peroxidase gene is expressed in the eosinophilic subline of human HL-60 cells but not in the neutrophilic subline or in parental HL-60 cells.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the Bacillus stearothermophilus peroxidase gene (perA).

The gene encoding a thermostable peroxidase was cloned from the chromosomal DNA of Bacillus stearothermophilus IAM11001 in Escherichia coli. The nucleotide sequence of the 3.1-kilobase EcoRI fragment containing the peroxidase gene (perA) and its flanking region was determined. A 2,193-base-pair open reading frame encoding a peroxidase of 731 amino acid residues (Mr, 82,963) was observed. A Shine-Dalgarno sequence was found 9 base pairs upstream from the translational starting site. The deduced amino acid sequence coincides with those of the amino terminus and four peptides derived from the purified peroxidase of B. stearothermophilus IAM11001. E. coli harboring a recombinant plasmid containing perA produced a large amount of thermostable peroxidase which comigrated on polyacrylamide gel electrophoresis with the B. stearothermophilus peroxidase. The peroxidase of B. stearothermophilus showed 48% homology in the amino acid sequence to the catalase-peroxidase of E. coli.     

Catalytic Profile of Arabidopsis Peroxidases,AtPrx-2, 25 and 71, Contributing to Stem Lignification

Lignins are aromatic heteropolymers that arise from oxidative coupling of lignin precursors, including lignin monomers (p-coumaryl, coniferyl, and sinapyl alcohols), oligomers, and polymers. Whereas plant peroxidases have been shown to catalyze oxidative coupling of monolignols, the oxidation activity of well-studied plant peroxidases, such as horseradish peroxidase C (HRP-C) and AtPrx53, are quite low for sinapyl alcohol. This characteristic difference has led to controversy regarding the oxidation mechanism of sinapyl alcohol and lignin oligomers and polymers by plant peroxidases. The present study explored the oxidation activities of three plant peroxidases, AtPrx2, AtPrx25, and AtPrx71, which have been already shown to be involved in lignification in the Arabidopsis stem. Recombinant proteins of these peroxidases (rAtPrxs) were produced in Escherichia coli as inclusion bodies and successfully refolded to yield their active forms. rAtPrx2, rAtPrx25, and rAtPrx71 were found to oxidize two syringyl compounds (2,6-dimethoxyphenol and syringaldazine), which were employed here as model monolignol compounds, with higher specific activities than HRP-C and rAtPrx53. Interestingly, rAtPrx2 and rAtPrx71 oxidized syringyl compounds more efficiently than guaiacol. Moreover, assays with ferrocytochrome c as a substrate showed that AtPrx2, AtPrx25, and AtPrx71 possessed the ability to oxidize large molecules. This characteristic may originate in a protein radical. These results suggest that the plant peroxidases responsible for lignin polymerization are able to directly oxidize all lignin precursors.     

豌豆卷须肌动蛋白Ⅱ类异型体cDNA克隆的序列分析

分析１５个豌豆卷须肌动蛋白ｃＤＮＡ 克隆的限制性内切酶图谱,发现在豌豆卷须中至少存在三类肌动蛋白异型体,分别命名为Ⅰ类（ＰＥＡｃ Ⅰ）、Ⅱ类（ＰＥＡｃ Ⅱ）和Ⅲ类（ＰＥＡｃ Ⅲ）异型体．三类异型体的克隆数目分别为１０、４和１个,表明三类异型体在豌豆卷须中的表达是不同的,很可能具有组织或发育阶段的特异性．对Ⅱ类异型体的三个ｃＤＮＡ 克隆ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１进行了全序列测定,所测定的序列已被ＧｅｎＢａｎｋ 数据库所接受．测序结果表明,ＰＥＡｃ３、ＰＥＡｃ９和ＰＥＡｃ１１的序列长度分别为１５５０、１６８０和１０９１个核苷酸,其中编码区长１１３４个核苷酸,编码的氨基酸长度为３７７（ＰＥＡｃ１１缺少编码氨基端前９６个氨基酸的核苷酸序列）．三个克隆的核苷酸序列完全相同,差别仅在于３′非翻译区的长度不同,即ｐｏｌｙ（Ａ）的加入位点不同．这说明它们可能是由同一基因转录而来,但转录后的加工过程不同．豌豆卷须中肌动蛋白基因ｐｏｌｙ（Ａ）加入位点的使用,可能与组织或发育的特异性表达有关．此外,豌豆卷须肌动蛋白三类异型体之间的核苷酸序列同源性为８０％ ,氨基酸序列同源性为９４％ ．     

地衣芽孢杆菌α-淀粉酶基因的克隆和及其启动子功能鉴定

根据已知α-淀粉酶编码基因保守区核苷酸序列,通过PCR和反向PCR技术克隆出Bacillus licheniformisCICIM B0204α-淀粉酶编码基因amyL全长序列及其上下游序列。B.licheniformisCICIM B0204amyL由1539bp组成,其上游180bp为启动子序列,下游160bp为终止子序列;成熟肽由512个氨基酸残基组成,氨基端的29个氨基酸残基为α-淀粉酶的信号肽。通过基因及其氨基酸序列比对发现,amyL及其编码产物与芽孢杆菌来源的α-淀粉酶具有高度相似性。将amyL的结构基因在PT7介导下于大肠杆菌中诱导表达,获得具有α-淀粉酶活性的表达产物。将amyL的启动子序列和信号肽序列与B.licheniformisCICIM B2004的β-甘露聚糖酶结构基因进行读框内重组,在大肠杆菌中获得了β-甘露聚糖酶的分泌表达,重组大肠杆菌表达295U/mL的β-甘露聚糖酶酶活。     

PPARGC1A基因Thr394Thr/Gly482Ser多态性与2型糖尿病的关联研究

对344例2型糖尿病患者和307名正常人的PPARGC1A基因单核苷酸多态性rs2970847(Thr394Thr)和rs8192678(Gly482Ser)与2型糖尿病的关系进行了单标记和单体型关联分析以及Logistic回归分析。在单标记分析中,对照组与病例组Thr394Thr的基因型和等位基因频率有显著差异(基因型, P =0.006; 等位基因, P < 0.001); Logistic回归和单体型分析表明, Thr394Thr的AA基因型及Thr394(ACA)-Ser482单体型增加患2型糖尿病的风险。Gly482Ser的基因型和等位基因频率在对照组与病例组间无显著差异。PPARGC1A基因是湖北汉人的一个2型糖尿病易感基因。