Characterization and sequence of a novel nitrate reductase from barley

Summary Barley (Hordeum vulgare L.) has both NADH-specific and NAD(P)H-bispecific nitrate reductases. Genomic and cDNA clones of the NADH nitrate reductase have been sequenced. In this study, a genomic clone (pMJ4.1) of a second type of nitrate reductase was isolated from barley by homology to a partial-length NADH nitrate reductase cDNA and the sequence determined. The open reading frame encodes a polypeptide of 891 amino acids and its interrupted by two small introns. The deduced amino acid sequence has 70% identity to the barley NADH-specific nitrate reductase. The non-coding regions of the pMJ4.1 gene have low homology (ca. 40%) to the corresponding regions of the NADH nitrate reductase gene. Expression of the pMJ4.1 nitrate reductase gene is induced by nitrate in root tissues which corresponds to the induction of NAD(P)H nitrate reductase activity. The pMJ4.1 nitrate reductase gene is sufficiently different from all previously reported higher plant nitrate reductase genes to suggest that it encodes the barley NAD(P)H-bispecific nitrate reductase.Scientific Paper No. 9101-14. College of Agriculture and Home Economics Research Center, Washington State University, Research Project Nos. 0233 and 0745     

cDNA cloning,sequence identification and tissue expression distribution of three novel porcine genes: UCHL3, RIT1 and CCND3

The complete CDS sequences of three porcine genes: UCHL3, RIT1 and CCND3 were amplified using RT-PCR based on the sequence information of the mouse or other mammals and referenced highly homologous pig ESTs. Sequence analysis of these three genes revealed that the porcine UCHL3 gene encodes a protein of 230 amino acids and has high homology with the ubiquitin carboxyl-terminal hydrolase isozyme L3 (UCHL3) of four species-bovine (97%), human (96%), mouse (95%) and rat (94%). The porcine RIT1 gene encodes a protein of 219 amino acids and has high homology with the GTP-binding protein Rit1 (RIT1) of two species-human (97%), mouse (97%). The porcine CCND3 gene encodes a protein of 292 amino acids and has high homology with the G1/S-specific cyclin-D3 (CCND3) of four species-bovine (98%), human (97%), mouse (93%) and rat (92%). The phylogenetic tree analysis revealed that the swine UCHL3 has a closer genetic relationship with the UCHL3 of bovine, and the swine RIT1 has closer genetic relationships with the RIT1 of human, but the swine CCND3 has a closer genetic relationship with the CCND3 of bovine. The RT-PCR gene expression analysis indicated that the swine UCHL3, RIT1 and CCND3 genes were differentially expressed in tissues including small intestine, large intestine, liver, muscle, fat, lung, spleen and kidney. Our experiment established the primary foundation for further research on these three swine genes.     

Cloning and expression of a gene encoding a root specific nitrate reductase in bean (Phaseolus vulgaris)

A structural gene encoding nitrate reductase (NR) in the legume Phaseolus vulgaris cv. Saxonia has been cloned and sequenced. The NR gene encodes a protein of 881 amino acids with a MW of 99.2 kDa. The coding sequence is interrupted by three introns, which are located in the molybdopterin cofactor binding domain. In the flanking regions the signals of a functional eukaryotic gene are present. The gene is the smallest NR gene so far identified in higher plants. Comparison to other NRs shows homology ranging from 65 to 74% at the amino acid level. The similarity is highest for the three functional domains, and lowest in the N-terminal end of the protein. mRNA studies demonstrate that the gene is nitrate inducible and expressed exclusively in the roots of bean. Southern blot analysis indicates the presence of a second NR gene in bean. This gene may encode a NR enzyme expressed in leaves.     

Crystal structure of vestitone reductase from alfalfa (Medicago sativa L.)

Isoflavonoids are commonly found in leguminous plants, where they play important roles in plant defense and have significant health benefits for animals and humans. Vestitone reductase catalyzes a stereospecific NADPH-dependent reduction of (3R)-vestitone in the biosynthesis of the antimicrobial isoflavonoid phytoalexin medicarpin. The crystal structure of alfalfa (Medicago sativa L.) vestitone reductase has been determined at 1.4 A resolution. The structure contains a classic Rossmann fold domain in the N terminus and a small C-terminal domain. Sequence and structural analysis showed that vestitone reductase is a member of the short-chain dehydrogenase/reductase (SDR) superfamily despite the low levels of sequence identity, and the prominent structural differences from other SDR enzymes with known structures. The putative binding sites for the co-factor NADPH and the substrate (3R)-vestitone were defined and located in a large cleft formed between the N and C-terminal domains of enzyme. Potential key residues for enzyme activity were also identified, including the catalytic triad Ser129-Tyr164-Lys168. A molecular docking study showed that (3R)-vestitone, but not the (3S) isomer, forms favored interactions with the co-factor and catalytic triad, thus providing an explanation for the enzyme's strict substrate stereo-specificity.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

Analysis of β-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrifleans, and their expression in Escherichia coli

Abstract The β-ketothiolase gene ( phaA ) and acetoacetyl-CoA reductase gene ( phaB ) were isolated from Paracoccus denitrificans . Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40744 Da and of 242 amino acids with a molecular mass of 25614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4–74.0% for the phaA gene product and 47.6–80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli . In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA - phaB , in P. denitrificans . NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.     

Trypanothione reductase of Trypanosoma congolense: gene isolation, primary sequence determination, and comparison to glutathione reductase

The gene encoding trypanothione reductase, the redox disulfide-containing flavoenzyme that is unique to the parasitic trypanosomatids (Shames et al., 1986), has been isolated from the cattle pathogen Trypanosoma congolense. Library screening was carried out with inosine-containing oligonucleotide probes encoding sequences determined from two active site peptides isolated from the purified Crithidia fasciculata enzyme. The nucleotide sequence of the gene was determined according to the dideoxy chain termination method of Sanger. The structural gene is 1476 nucleotides long and encodes 492 amino acids. We have identified the active site peptide containing the redox-active disulfide, a peptide corresponding to the histidine-467 region of human erythrocyte glutathione reductase, as well as the flavin binding domain that is highly conserved in all disulfide-containing flavoprotein reductase enzymes. Alignment of five tryptic peptides (80 residues) isolated from the C. fasciculata trypanothione reductase with the primary sequence of the T. congolense enzyme showed 88% homology with 76% identity. Additionally, a sequence comparison of the glutathione reductase from Escherichia coli or human erythrocytes to T. congolense trypanothione reductase reveals greater than 50% homology. A search for the amino acid residues in the primary sequence of trypanothione reductase functionally active in binding/catalysis in human erythrocyte glutathione reductase shows that only the two arginine residues (Arg-37 and Arg-347), shown by X-ray crystallographic data to hydrogen bond to the GS1 glutathione glycyl carboxylate, are absent.     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM 109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) fromβ-hydroxyacetophenone without any additive to regenerate NAD+ from NADH.     

The DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the yeast ubiquinol-cytochrome c reductase: a protein with an extremely high content of acidic amino acids

We have determined the DNA sequence of the nuclear gene coding for the 17-kd subunit VI of the ubiquinol-cytochrome c reductase. The reading frame found encodes a putative polypeptide of 17 394 daltons. This protein is highly unusual: 38% of its residues are acidic and 14% are basic amino acids. The most notable feature in the protein sequence is a stretch of 25 consecutive acidic amino acids. The polypeptide has homology with the 9-kd so-called 'hinge' protein of beef-heart complex III, which also has a cluster of acidic residues. Acidic amino acids are likely to be essential for the function of these proteins, since their degree of conservation is higher than that of other residues.     

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.     

大豆查尔酮还原酶基因CHR1的功能研究

为了验证查尔酮还原酶基因CHR1在大豆苷元合成中的作用, 文章克隆了CHR1基因并构建了RNA干扰表达载体pCPB-CHR1-RNAi, 将载体转化受体大豆品种“吉农28”中, 以期抑制CHR1基因的转录。通过农杆菌介导的遗传转化和PCR检测得到4株T₀ 代阳性植株, 13株T₁代阳性植株。Southern blotting结果表明, 功能元件以单拷贝的形式整合到大豆的基因组中。利用实时荧光定量PCR法(Quantitative real-time PCR, qRT-PCR)测定CHR1基因在mRNA水平上的表达量, 结果表明, 转基因大豆植株中CHR1的表达量与未转化的受体大豆相比降低了60%~99%; 高效液相色谱法(High performance liquid chromatography, HPLC)检测到合成大豆苷元过程中的前体物质异甘草素的含量降低了38.7%。该RNA干扰机制在转录水平上抑制了CHR1基因的表达。     

Identification and isolation of the gene encoding the small subunit of ribonucleotide reductase from Saccharomyces cerevisiae: DNA damage-inducible gene required for mitotic viability.

Ribonucleotide reductase catalyzes the first step in the pathway for the production of deoxyribonucleotides needed for DNA synthesis. The gene encoding the small subunit of ribonucleotide reductase was isolated from a Saccharomyces cerevisiae genomic DNA expression library in lambda gt11 by a fortuitous cross-reaction with anti-RecA antibodies. The cross-reaction was due to an identity between the last four amino acids of each protein. The gene has been named RNR2 and is centromere linked on chromosome X. The nucleotide sequence was determined, and the deduced amino acid sequence, 399 amino acids, shows extensive homology with other eucaryotic ribonucleotide reductases. Transplason mutagenesis was used to disrupt the RNR2 gene. A novel assay using colony color sectoring was developed to demonstrate visually that RNR2 is essential for mitotic viability. RNR2 encodes a 1.5-kilobase mRNA whose levels increase 18-fold after treatment with the DNA-damaging agent 4-nitroquinoline 1-oxide. CDC8 was also found to be inducible by DNA damage, but POL1 and URA3 were not inducible by 4-nitroquinoline 1-oxide. The expression of these genes defines a new mode of regulation for enzymes involved in DNA biosynthesis and sharpens our picture of the events leading to DNA repair in eucaryotic cells.