Molecular Cloning and Evolutionary Analysis of Hemoglobin α-Chain Genes in Bats

Bats are the only mammals with the capacity for powered flight. When flying, they need abundant energy and oxygen. According to previous works, the hemoglobin (Hb) oxygen loading function of bats is insensitive to variations in body temperature, although different bat species have different heat sensitivity. We cloned Hb α-chain sequences from eight bat species to investigate whether they have different characteristics. We found that Hb in the bat lineages is under purifying selection, which accords with the importance of its function in bats. Three turn regions in bat Hb, however, have distinct evolutionary rates compared with those of other mammals, and the codons in these regions have an accelerated rate of evolution. These codons are under divergent selection in bats. These changes in Hb may have occurred in response to the physiological requirements of the species concerned, as adaptations to different lifestyles.     

Molecular Cloning and Crystal Structural Analysis of a Novel β-N-Acetylhexosaminidase from Paenibacillus sp. TS12 Capable of Degrading Glycosphingolipids

We report the molecular cloning and characterization of two novel β-N-acetylhexosaminidases (β-HEX, EC 3.2.1.52) from Paenibacillus sp. strain TS12. The two β-HEXs (Hex1 and Hex2) were 70% identical in primary structure, and the N-terminal region of both enzymes showed significant similarity with β-HEXs belonging to glycoside hydrolase family 20 (GH20). Interestingly, however, the C-terminal region of Hex1 and Hex2 shared no sequence similarity with the GH20 β-HEXs or other known proteins. Both recombinant enzymes, expressed in Escherichia coli BL21(DE3), hydrolyzed the β-N-acetylhexosamine linkage of chitooligosaccharides and glycosphingolipids such as asialo GM2 and Gb4Cer in the absence of detergent. However, the enzyme was not able to hydrolyze GM2 ganglioside in the presence or in the absence of detergent. We determined three crystal structures of Hex1; the Hex1 deletion mutant Hex1-ΔC at a resolution of 1.8 Å; Hex1-ΔC in complex with β-N-acetylglucosamine at 1.6 Å; and Hex1-ΔC in complex with β-N-acetylgalactosamine at 1.9 Å. We made a docking model of Hex1-ΔC with GM2 oligosaccharide, revealing that the sialic acid residue of GM2 could hinder access of the substrate to the active site cavity. This is the first report describing the molecular cloning, characterization and X-ray structure of a procaryotic β-HEX capable of hydrolyzing glycosphingolipids.     

Anti-HIV Ⅰ/Ⅱ Activity and Molecular Cloning of a Novel Mannose/Sialic Acidbinding Lectin from Rhizome of Polygonatum cyrtonema Hua

The anti-human immunodeficiency virus （HIV） Ⅰ/Ⅱ activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins （MBLs）. The anti-HIV activity of Polygonatum cyrtonema Hua lectin （PCL） was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3＇/5＇-rapid amplification of cDNA ends （RACE）, the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site （QDNVY） was found in subdomain Ⅰ of PCL, that of subdomain Ⅱ and Ⅲ changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region （TIR） in mRNA of PCL benefits its high expression in rhizomes.     

A Comparative Structure–Function Analysis and Molecular Mechanism of Action of Endonucleases from Serratia marcescens and Physarum polycephalum

Structural and functional characteristics were compared for wild-type nuclease from Serratia marcescens, which belongs to the family of DNA/RNA nonspecific endonucleases, its mutational forms, and the nuclease I-PpoI from Physarum polycephalum, which is a representative of the Cys-His box-containing subgroup of the superfamily of extremely specific intron-encoded homing DNases. Despite the lack of sequence homology and the overall different topology of the Serratia marcescens and I-PpoI nucleases, their active sites have a remarkable structural similarity. Both of them have a unique magnesium atom in the active site, which is a part of the coordinatively bonded water–magnesium complex involved in their catalytic acts. In the enzyme–substrate complexes, the Mg²⁺ ion is chelated by an Asp residue, coordinates two oxygen atoms of DNA, and stabilizes the transition state of the phosphate anion and 3"-OH group of the leaving nucleotide. A new mechanism of the phosphodiester bond cleavage, which is common for the Serratia marcescens and I-PpoI nucleases and differs from the known functioning mechanism of the restriction and homing endonucleases, was proposed. It presumes a His residue as a general base for the activation of a non-cluster water molecule at the nucleophilic in line displacement of the 3"-leaving group. A strained metalloenzyme–substrate complex is formed during hydrolysis and relaxes to the initial state after the reaction.     

Cloning and Expression of Low Molecular Weight Glutenin Genes from the Chinese Elite Wheat Cultivar ＂Xiaoyan 54＂

The low molecular weight （LMW） glutenln subunlts account for 40% of wheat gluten protein content by mass and these proteins are considered to significantly affect dough quality characteristics. Five new full-length LMW glutenln genes （designated LMW-5, LMW-7, LMW-42, LMW-58, and LMW-34） were isolated from the Chinese elite wheat cultivar ＂Xlaoyan 54＂ by PCR amplification of genomlc DNA using a pair of degenerate primers designed from the conserved sequences of the N- and C-terminal regions of published LMW glutenln genes. Deduced amino acid sequence analysis showed that LMW-5 belongs to the LMW-i type genes and that the other four belong to LMW-m type genes. Sequence comparisons revealed that point mutations occasionally occurred in signal peptide and N-terminus domains and often existed in domain III and domain V. Small insertions and deletions are represented in the repetitive domain. There is a stop codon after amino acid position 110 In the repetitive domain of LMW.34, indicating that It is a pseudogene. The other four genes have complete open reading frames and the putative mature regions of these genes were subcloned Into pET-30a expression vector and successfully expressed in Escherlchla coll. Protein sodium dodecyl sulfate-polyacrylamlde gel electro- phoresls analysis showed that all proteins expressed in E. coil by the four genes could be related to B-group LMW glutenln subunits of wheat.     

Molecular Cloning and Sequence Analysis of PGC-1α cDNA in Tibetan Antelope

以藏羚羊(Pantholops hodgsonii)及同海拔分布的藏系绵羊(Tibetan Sheep)的心肌组织为材料,提取总RNA,利用逆转录聚合酶链反应(RT-PCR)技术扩增出过氧化物酶体增生物激活受体γ辅激活因子-1α(PGC-1α)的基因编码区cDNA片段,与载体连接构建重组质粒,经转化、扩增培养、鉴定后测序.利用生物信息学方法分析显示,藏羚羊和藏系绵羊的PGC-1α基因编码区长度均为2 349 bp,编码797个氨基酸(GenBank登录号分别为:JF449959和JF449960);与其他脊椎动物PGC-1α基因的核苷酸及氨基酸序列相似性达到90％以上;其包含RNA/DNA结合位点、RNA识别基序(RRM)、与核呼吸因子1( NRF-1)及肌细胞增强因子2C(MEF2C)相互作用的区域、富含丝氨酸/精氨酸的结构域、负调节功能结构域、LXXLL模体以及TPPTTPP和DHDYCQ两个保守序列,14个氨基酸差异性位点位于以上部分功能结构域中;此外,磷酸化位点的预测提示藏羚羊可能存在一个潜在的蛋白激酶G的磷酸化位点(第329位的苏氨酸).本研究成功克隆出了藏羚羊PGC-1α基因的编码区序列,为从能量代谢角度深入探讨藏羚羊适应高原的分子生物学机制提供了新的思路.     

Molecular Cloning and Characterization of γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694

γ-Glutamyltranspeptidase from Pseudomonas nitroreducens IFO12694 (PnGGT) exhibited higher hydrolytic activity than transfer activity, as compared with other γ-glutamyltranspeptidases (GGTs). PnGGT showed little activity towards most of L-amino acids and towards glycyl-glycine, which is often used as a standard γ-glutamyl accepter in GGT transfer reactions. The preferred substrates for PnGGT as a γ-glutamyl accepter were amines such as methylamine, ethylamine, and isopropylamine.     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.     

Molecular Cloning and Characterization of Enolase from Oilseed Rape （Brassica napus）

An enolase-encoding cDNA clone in oilseed rape (Brassica napus L.) was isolated. This gene (accession number: AY307449) had a total length of 1 624 bp with an open reading frame of 1 335 bp, and encoded a predicted polypeptide of 444 amino acids with a molecular weight of 47.38 kD. The deduced amino acid sequence shared identity with a number of enolases ranging from Bacillus subtilis to human beings and had much higher identity with other plant enolases than with enolases from Bacillus, yeast and human beings. Comparison of its primary structure with those of other enolases revealed the presence of an insertion of five amino acids in enolase of B. napus. Southern blotting analysis of genomic DNA indicated that enolase was likely to be a low-copy gene in the oilseed rape genome. Expression of the cloned enolase gene increased under salt stress, but decreased in response to low temperature. Our studies suggested that the cloned gene was a new member of plant enolase gene family, which contributed to the energy supply in stress-treated tissues.     

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb （Amaryllidaceae）

A novel mannose-bindlng aggiutinln was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% （NH4）2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel flitration on Sephscryl S-100. The purified Z. candida agglutlnln （ZCA） migrated as a single band of 12 kDa on sodium dodecyi suifate-poiyecryiamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the iectln, as datermlned by gel filtration chromatography, was 48 kDa. The results Indicated that ZCA was composed of four Identical subunlts of 12 kDa each （homotetramerlc nature）. The ZCA agglutlhated rabbit erythrocytes, Escherichla coil and Saccharomyces cerevislae ceils at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bloassays Indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was 〉 90% at 0.26%. A degenerate primer was designed In accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3＇- and 5＇-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA Includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptlde, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA Indicated that Its three-dimensional atructure strongly resembies that of the snowdrop aggiutinin. Blocks＇ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes （QDNY）.     

Molecular Cloning of α-Amylase Genes from DROSOPHILA MELANOGASTER. I. Clone Isolation by Use of a Mouse Probe

A cloned alpha-amylase cDNA sequence from the mouse is homologous to a small set of DNA sequences from Drosophila melanogaster under appropriate conditions of hybridization. A number of recombinant lambda phage that carry homologous Drosophila genomic DNA sequences were isolated using the mouse clone as a hybridization probe. Putative amylase clones hybridized in situ to one or the other of two distinct sites in polytene chromosome 2R and were assigned to one of two classes, A and B. Clone lambda Dm32, representing class A, hybridizes within chromosome section 53CD. Clone lambda Dm65 of class B hybridizes within section 54A1-B1. Clone lambda Dm65 is homologous to a 1450- to 1500-nucleotide RNA species, which is sufficiently long to code for alpha-amylase. No RNA homologous to lambda Dm32 was detected. We suggest that the class B clone, lambda Dm65, contains the functional Amy structural gene(s) and that class A clones contain an amylase pseudogene.     

Comparative and evolutionary analysis of new variants of ω-gliadin genes from three A-genome diploid wheats

A genomic polymerase chain reaction (PCR) cloning strategy was applied to isolate ω-gliadin sequences from three A-genome diploid wheats (Triticum monococcum, T. boeoticum and T. urartu). Amplicon lengths varied from 744 and 1,044 bp, and those of the corresponding deduced mature proteins from 248 to 348 residues. The primary structure of the deduced polypeptides comprised a short N- and C-terminal conserved domain, and a long, variable repetitive domain. A phylogenetic analysis recognised several clades: the first consisted of three T. aestivum sequences; the second and the third two T. boeoticum and six T. monococcum sequences; and the rest four T. urartu and three T. aestivum sequences. Among the functional (non-pseudogene) ARQ/E-type ω-gliadin sequences, two were derived from T. boeoticum and three from T. monococcum; one of the latter sequences appeared to be a chimera originating via illegitimate recombination between the other two T. monococcum sequences. None of the 12 intact ω-gliadin sequences contained any cysteine or methionine residues. We discussed the variation and evolution of A-genome ω-gliadin genes.     

Identification and Molecular Characterization of a Novel Type of α-galactosidase from Pyrococcus furiosus

An α-galactosidase gene from Pyrococcus furiosus was identified, cloned and functionally expressed in Escherichia coli. It is the first α-galactosidase from a hyperthermophilic archaeon described to date. The gene encodes a unique amino acid sequence compared to other α-galactosidases. Highest homology was found with α-amylases classified in family 57 of glycoside hydrolases. The 364 amino acid protein had a calculated mass of 41.6 kDa. The recombinant α-galactosidase specifically catalyzed the hydrolysis of para-nitrophenyl-α-galactopyranoside, and to some extent that of melibiose and raffinose. The enzyme proved to be an extremely thermo-active and thermostable α-galactosidase with a temperature optimum of 115°C and a half-life time of 15 hours at 100°C. The pH optimum is between 5.0 and 5.5. Sequence analysis showed four conserved carboxylic residues. Site-directed mutagenesis was applied to identify the potential catalytic residues. Glu117Ala showed decreased enzyme activity, which could be rescued by the addition of azide or formate. It is concluded that glutamate 117 is the catalytic nucleophile, whereas the acid/base catalyst remains to be identified.     

Comparative Analysis of Polymorphous Genes of Glutathione-S-Transferases in North-Western Populations of Russia—Healthy and Patients with Bronchial Asthma

The ratio between polymorphous variants of two glutathione S-transferase (M1 and T1) genes (GSTM1 and GSTT1) was studied in healthy people and patients with bronchial asthma (BA) from northwestern Russia. The relative risk of development of BA amounted to 8.5 (C1 95%: 3.623–10.956) in people with GSTM1 0/0,GSTT1 0/0 genotype. No statistically significant differences in distribution of GSTM1 and GSTT1 genotypes were revealed between male and female patients with BA as was among patients with different severity of the disease. Frequency of homozygous GSTM1 0/0 and GSTT1 0/0 differed between patients with the beginning of the disease at the age before and after 30 years. The relative risk of BA development with GSTM1 0/0, GSTT1 0/0 genotype was increased 9.6 times (CI 95%: 3.90–23.85) in patients younger than 30 years, while 3.6 times (CI 95%: 2.15–5.98), in patients older than 30 years.     

The Family of Peanut Fatty Acid Desaturase Genes and a Functional Analysis of Four ω-3 AhFAD3 Members

Plant Molecular Biology Reporter - The synthesis of α-linolenic acid (ALA) requires the activity of ω-3 fatty acid desaturases (ω-3 FADs). The quality of peanut oil would be much...     

Purification,Cloning, Characterization,and N-Glycosylation Analysis of a Novel β-Fructosidase from Aspergillus oryzae FS4 Synthesizing Levan- and Neolevan-Type Fructooligosaccharides

β-Fructosidases are a widespread group of enzymes that catalyze the hydrolysis of terminal fructosyl units from various substrates. These enzymes also exhibit transglycosylation activity when they function with high concentrations of sucrose, which is used to synthesize fructooligosaccharides (FOS) in the food industry. A β-fructosidase (BfrA) with high transglycosylation activity was purified from Aspergillus oryzae FS4 as a monomeric glycoprotein. Compared with the most extensively studied Aspergillus spp. fructosidases that synthesize inulin-type β-(2-1)-linked FOS, BfrA has unique transfructosylating property of synthesizing levan- and neolevan-type β-(2-6)-linked FOS. The coding sequence (bfrAFS4, 1.86 kb) of BfrA was amplified and expressed in Escherichia coli and Pichia pastoris. Both native and recombinant proteins showed transfructosylation and hydrolyzation activities with broad substrate specificity. These proteins could hydrolyze the following linkages: Glc α-1, 2-β Fru; Glc α-1, 3-α Fru; and Glc α-1, 5-β Fru. Compared with the unglycosylated E. coli-expressed BfrA (E.BfrA), the N-glycosylated native (N.BfrA) and the P. pastoris-expressed BfrA (P.BfrA) were highly stable at a wide pH range (pH 4 to 11), and significantly more thermostable at temperatures up to 50°C with a maximum activity at 55°C. Using sucrose as substrate, the Km and k_cat values for total activity were 37.19±5.28 mM and 1.0016±0.039×10⁴ s⁻¹ for N.BfrA. Moreover, 10 of 13 putative N-glycosylation sites were glycosylated on N.BfrA, and N-glycosylation was essential for enzyme thermal stability and optima activity. Thus, BfrA has demonstrated as a well-characterized A. oryzae fructosidase with unique transfructosylating capability of synthesizing levan- and neolevan-type FOS.