褐飞虱乙酰胆碱酯酶基因全长cDNA的克隆及序列分析

利用反转录聚合酶链式反应(RT_PCR)结合快速扩增cDNA末端(RACE)技术克隆了褐飞虱Nilaparvata lugens 乙酰胆碱酯酶基因cDNA。该cDNA全长2 467 bp,包含一个1 938 bp的开放阅读框(GenBank登录号AJ852420); 在推导出的646个氨基酸残基的前体蛋白中, N端的前30个氨基酸残基为信号肽,随后的616个氨基酸残基是成熟的乙酰胆碱酯酶序列,其预测的分子量为69 418 D。在一级结构中,形成催化活性中心的3个氨基酸残基(Ser242,Glu371和His485),以及在亚基内形成二硫键的6个半胱氨酸完全保守; 位于催化功能域的14个芳香族氨基酸中有10 个完全保守。该酶的氨基酸序列与黑尾叶蝉的同源性最高,达83%。对来自23种昆虫（包括褐飞虱）的30个乙酰胆碱酯酶的聚类分析显示,褐飞虱的乙酰胆碱酯酶与其中6个Ⅱ型乙酰胆碱酯酶（AChE2）同属一个支系; 此外,只存在于昆虫AChE2中的超变区及特异的氨基酸残基,也存在于褐飞虱的乙酰胆碱酯酶中。以上结果表明,所克隆的褐飞虱的乙酰胆碱酯酶基因是一个与黑腹果蝇的orthologous型基因同源的AChE2基因。     

Bovine brain acetylcholinesterase primary sequence involved in intersubunit disulfide linkages

Three distinct classes of membrane-bound acetylcholinesterases (AChEs) have been identified. A12 AChE is composed of 12 catalytic subunits that are linked to noncatalytic collagen-like subunits through intersubunit disulfide bonds. G2 AChE is localized in membranes by a glycoinositol phospholipid covalently linked to the C-terminal amino acid. Brain G4 AChE involves two catalytic subunits linked by a direct intersubunit disulfide bond while the other two are disulfide-linked to a membrane-binding 20-kDa noncatalytic subunit. Molecular cloning studies have so far failed to find evidence of more than one AChE gene in any organism although alternative splicing of torpedo AChE mRNA results in different C-terminal sequences for the A12 and G2 AChE forms. Support for a single bovine AChE gene is provided in this report by amino acid sequencing of the N-terminal domains from the G2 erythrocyte, G4 fetal serum, and G4 brain AChE. Comparison of the 38-amino acid sequences reveals virtually complete identity among the three AChE forms. Additional extensive identity between the fetal serum and brain AChEs was demonstrated by sequencing several brain AChE peptides isolated by high performance liquid chromatography after trypsin digestion of nitrocellulose blots of brain AChE catalytic subunits. Cysteines involved in intersubunit disulfide linkages in brain AChE were reduced selectively with dithiothreitol in the absence of denaturants and radioalkylated with iodoacetamide. The observed sequence of the major radiolabeled tryptic peptide was C*SDL, where C* was the radioalkylated cysteine residue. This sequence is precisely the same as that observed at the C terminus of fetal bovine serum AChE and shows close homology to the C-terminal sequence of torpedo A12 AChE. We conclude that the mammalian brain G4 AChEs utilize the same exon splicing pattern as the A12 AChEs and that factors other than the primary sequence of the AChE catalytic subunits dictate assembly with either the collagen-like or the 20-kDa noncatalytic subunits.     

Primary structure of acetylcholinesterase: implications for regulation and function

A cDNA encoding acetylcholinesterase (AChE) (EC 3.1.1.7) from Torpedo californica was isolated and from its nucleotide sequence the entire amino acid sequence of the processed protein and a portion of the leader peptide has been deduced. Approximately 70% of the tryptic peptides from the catalytic subunit of the 11 S form have been sequenced, and a comparison of the peptide sequences with the sequence inferred from the cDNA suggests that the cDNA sequence derives from mRNA for the 11 S form of the enzyme. The amino acid sequence is preceded by a hydrophobic leader peptide and contains an open reading frame encoding for 575 amino acids characteristic of a secreted globular protein. Eight cysteines, most of which are disulfide linked, are found along with four potential sites of N-linked glycosylation. The active-site serine is located at residue 200. Local homology is found with other serine hydrolases in the vicinity of the active site, but the enzyme shows striking global homology with the COOH-terminal portion of thyroglobulin. Further comparison of the amino acid sequences of the individual enzyme forms with other cDNA clones that have been isolated should resolve the molecular basis for polymorphism of the AChE species.     

Identification and characterization of a cDNA encoding the acetylcholinesterase of Haematobia irritans (L.) (Diptera: Muscidae).

A 2217-nucleotide cDNA presumptively encoding acetylcholinesterase (AChE) of the horn fly, Haematobia irritans (L.) was sequenced. The open reading frame (ORF) encoded a 91 amino acid secretion signal peptide and a 613 amino acid mature protein with 95% identity and 98% similarity to the AChE of Musca domestica (L.). Structural features characteristic of the M. domestica and Drosophila melanogaster AChEs are conserved in the H. irritans AChE. The M. domestica and D. melanogaster AChEs are target sites for organophosphate inhibition as previously shown (Walsh et al. 2001. Biochem. J. 359: 175-181, Kozaki et al. 2002. Appl. Entomol. Zool. 37: 213-218), suggesting that this H. irritans AChE2 may be the target site for organophosphate.     

Two different genes encoding acetylcholinesterase existing in cotton aphid (Aphis gossypii).

Two acetylcholinesterase (AChE) genes, Ace1 and Ace2, have been cloned from cotton aphid, Aphis gossypii Glover, using the rapid amplification of cDNA ends (RACE) technique. To the best of our knowledge, this should be the first direct molecular evidence that multiple AChE genes exist in insects. The Ace1 gene was successfully amplified along its full length of 2371 bp. The open reading frame is 2031 bp long and encodes 676 amino acids (GenBank accession No. AF502082). The Ace2 gene was amplified as a mega-fragment of 2130 bp lacking part of 5'-end untranslated region (UTR). The open reading frame is 1992 bp long and ecodes a protein of 664 amino acids (GenBank accession No. AF502081). Both genes have the conserved amino acids and features shared by the AChE family, but share only 35% identity in amino acid sequence. The Ace1 gene is highly homologous to the AChE gene of Schizaphis graminum (AF321574) with 95% identity, and Ace2 to that of Myzus persicae (AF287291) with 92% identity. Phylogenetic analysis showed that the two cloned AChEs of A. gossypii are different in evolution. The phylogenetic tree generated by the PHYLIP program package inferred that AChE2 of A. gossypii is a more ancestral form of AChE. Homology modeling of structures using Torpedo californica (2ACE_) and Drosophila melanogaster (1Q09:A) native acetylcholinesterase structure as main template indicated that the two AChEs of Aphis gossypii might have different three-dimensional structures. Alternative splicing of Ace1 near the 5'-end resulting in two proteins differing by the presence or absence of a fragment of four amino acids is also reported.     

Sequence of a cDNA encoding acetylcholinesterase from susceptible and resistant two-spotted spider mite,Tetranychus urticae

Acetylcholinesterase (AChE) from two-spotted spider mites, Tetranychus urticae was compared between an organophosphate susceptible (TKD) and a resistant (NCN) strain. The AChE of TKD had lower affinity to acetylthiocholine and propionylthiocholine than that of NCN, and the inhibition of AChE by DDVP, ambenonium, eserine and n-methyl-eserine showed that NCN was more insensitive than TKD. AChE cDNA sequence was determined, and the 687 amino acids of primary structure were deduced. There were six replacements of amino acid residues in TKD and two in NCN. #F331(439)C was the only substitution unique to NCN, however, this mutation existed homozygously in only two out of nine mites. This residue is one of the gorge lining components, and #F331(439)C might act an important role in the sensitivity of AChE to the inhibitors.     

杀虫药剂抗性家蝇品系乙酰胆碱酯酶基因的特征分析

乙酰胆碱酯酶(AChE)是有机磷和氨基甲酸酯类杀虫药剂的作用靶标,这两大类杀虫药剂的广泛应用导致了昆虫对抗性的选择。靶标的修饰是某些昆虫产生抗性的分于机理,这种抗性是和AChE的变更型相关的,这些变更型的酶显示出对杀虫药剂的不被感性。利用RT-PCR和Streptavidin偶联磁珠技术从两种抗性家蝇(Musca domestica)品系D3和Kash中分别分离了AChE基因并测定了其按苷酸颅序。eDNA的可读框长2082bp．由此推导出了AChE的氨基酸顺序,通过与敏感家蝇品系Cooper的比较,发现了一些核苷酸顺序差异和4个氨基酸点突变,其中3个替代可能与杀虫药剂不敏感性有关。这一结果表明D3和Kash均属于CH₂抗性类型。     

Nucleotide sequence analysis of cDNA encoding the coat protein of cucumber mosaic virus: genome organization and molecular features of the protein

The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant.     

Characterization of trimeric acetylcholinesterase from a legume plant, <Emphasis Type="Italic">Macroptilium atropurpureum</Emphasis> Urb.

We recently identified plant acetylcholinesterases (E.C.3.1.1.7; AChEs) homologous to the AChE purified from a monocotyledon, maize, that are distinct from the animal AChE family. In this study, we purified, cloned and characterized an AChE from a dicotyledon, siratro. The full-length cDNA of siratro AChE is 1,441 nucleotides, encoding a 382-residue protein that includes a signal peptide. This AChE is a disulfide-linked 125-kDa homotrimer consisting of 41–42 kDa subunits, in contrast to the maize AChE, which exists as a mixture of disulfide and non-covalently linked 88-kDa homodimers. The plant AChEs apparently consist of various quaternary structures, depending on the plant species, similar to the animal AChEs. We compared the enzymatic properties of the dimeric maize and trimeric siratro AChEs. Similar to electric eel AChE, both plant AChEs hydrolyzed acetylthiocholine (or acetylcholine) and propionylthiocholine (or propionylcholine), but not butyrylthiocholine (or butyrylcholine), and their specificity constant was highest against acetylcholine. There was no significant difference between the enzymatic properties of trimeric and dimeric AChEs, although two plant AChEs had low substrate turnover numbers compared with electric eel AChE. The two plant AChE activities were not inhibited by excess substrate concentrations. Thus, similar to some plant AChEs, siratro and maize AChEs showed enzymatic properties of both animal AChE and animal BChE. On the other hand, both siratro and maize AChEs exhibited low sensitivity to the AChE-specific inhibitor neostigmine bromide, dissimilar to other plant AChEs. These differences in enzymatic properties of plant AChEs may reflect the phylogenetic evolution of AChEs. Kosuke Yamamoto and Yoshie S. Momonoki contributed equally to this work.     

Nucleotide sequence and characterization of a cDNA encoding the acetohydroxy acid isomeroreductase from Arabidopsis thaliana

The primary structure of acetohydroxy acid isomeroreductase from Arabidopsis thaliana was deduced from two overlapping cDNA. The full-length cDNA sequence predicts an amino acid sequence for the protein precursor of 591 residues including a putative transit peptide of 67 amino acids. Comparison of the A. thaliana and spinach acetohydroxy acid isomeroreductases reveals that the sequences are conserved in the mature protein regions, but divergent in the transit peptides and around their putative processing site.     

Cloning of rat brain protein kinase C complementary DNA

Four peptides derived from rat brain protein kinase C were partially sequenced. Using synthetic oligonucleotides deduced from the amino acid sequences as probes, a clone of complementary DNA (cDNA) was isolated from a cDNA library prepared from the same tissue. The nucleotide sequence of this cDNA clone revealed the primary structure of the carboxyl-terminal region as having 224 amino acids, with significant sequence homology with cyclic AMP-dependent and cyclic GMP-dependent protein kinases.     

Linkage analysis of an acetylcholinesterase gene in the house fly Musca domestica (Diptera: Muscidae)

Linkage of an acetylcholinesterase (AChE) gene was detected in the house fly, Musca domestica L., by using the backcross method between a strain, aabys, that had a morphological multichromosomal marker on each of the five autosomes and a wild strain, LPR. Both strains were homozygous in this gene, and we used eight single nucleotide polymorphisms (SNPs) between them to distinguish the parental sequences in the backcrossed progeny, two of which resulted in the amino acid substitiutions common to the Drosophila and Aedes AChEs insensitive to organophosphates and carbamates. F, appeared to be a wild phenotype, and the AChE gene was heterozyous of aabys and LPR. In the backcross progeny, 32 (2(5)) phenotypes appeared, and 10 phenotypes with one wild or morphological marker were picked up for genotyping by the SNPs of AChE gene. A combination of the morphological markers and the SNPs revealed that the AChE structural gene is linked to autosome 2 in the house fly.     

Mutations of acetylcholinesterase1 contribute to prothiofos-resistance in Plutella xylostella (L.)

Insensitive acetylcholinesterase (AChE) is involved in the resistance of organophosphorous and carbamate insecticides. We cloned a novel full-length AChE cDNA encoding ace1 gene from adult heads of the diamondback moth (DBM, Plutella xylostella). The ace1 gene encoding 679 amino acids has conserved motifs including catalytic triad, choline-binding site and acyl pocket. Northern blot analysis revealed that the ace1 gene was expressed much higher than the ace2 in all examined body parts. The biochemical properties of expressed AChEs showed substrate specificity for acetylthiocholine iodide and inhibitor specificity for BW284C51 and eserine. Three mutations of AChE1 (D229G, A298S, and G324A) were identified in the prothiofos-resistant strain, two of which (A298S and G324A) were expected to be involved in the prothiofos-resistance through three-dimensional modeling. In vitro functional expression of AChEs in Sf9 cells revealed that only resistant AChE1 is less inhibited with paraoxon, suggesting that resistant AChE1 is responsible for prothiofos-resistance.     

Mechanisms of monocrotophos resistance in cotton bollworm,Helicoverpa armigera (Hübner)

Insensitive acetylcholinesterase was identified as a resistance mechanism by comparing biochemical analysis with a laboratory selected monocrotophos resistant cotton bollworm (RR: 200) and the susceptible strain. The cDNA encoding AChE was cloned by the method of RACE (rapid amplification of cDNA ends). The complete AChE gene deduced from the cDNA consisted of a putative signal peptide of 32 amino acid residues, a mature protein of 615 residues, 5' untranslated regions (UTR) of 315 bp and 3' UTR of 324 bp. The coding sequence had a high degree of homology to the AChE from other insect species reported in the GenBank. After comparing analysis of the entire AChE gene sequence from 5 resistant and 6 susceptible cotton bollworm individuals, nine mutations were identified. One of them, the Ala/Thr mutation, is likely to be responsible for the AChE insensitivity to monocrotophos.     

Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications

Human liver fatty acid binding protein (L-FABP) cDNA clones were identified in a liver cDNA library. The two longest clones were completely sequenced. The nucleotide sequence predicts a protein of 127 amino acid residues. Identity of the clones was confirmed by limited amino acid sequence analysis of purified human L-FABP peptides and Edman degradation of radiolabeled in vitro translated FABP. Statistical analysis of the amino acid and mRNA sequences of human L-FABP, rat L-FABP, rat intestinal (I-) FABP, and mouse 422 protein indicates that the human and rat L-FABPs are highly homologous and that L-FABP and I-FABP diverged a long time ago (approximately 650-690 million years ago), although they are more closely related to each other than either of them is to 422 protein. Secondary structure predictions from the primary sequence of human and rat L-FABP reveal a region (residues 12-30) that might be the putative fatty acid binding domain of the two L-FABPs. Knowledge of the primary amino acid sequence of L-FABP and possible functional domains will be pivotal in further defining and understanding the mechanism of ligand binding and transfer by this protein.     

Comparative studies on the primary structure of acetylcholinesterases from bovine caudate nucleus and bovine erythrocytes

1. Comparison of partial amino acid sequences of G2-acetylcholinesterase (AChE) from bovine erythrocytes and G4-AChE from bovine caudate nucleus revealed no differences in primary structure between the two enzymes. The first 33 residues of the N-terminal sequences were identical. 2. In addition, the amino acid sequences of four peptides generated by tryptic and cyanogen bromide cleavage were identical for bovine erythrocyte and brain AChE, suggesting one identical major coding exon for the adult bovine AChE forms. Comparison of these sequences with that of fetal bovine serum AChE (Doctor et al., 1988), showed differences in residues 16, 181, 212, and 216. 3. Deglycosylation studies of the two adult enzyme forms revealed that the core protein of erythrocyte AChE has an approximately 4 kDa lower molecular mass than brain AChE. This most probably reflects differences in the C-terminal sequences of the two enzymes.     

UDP-glucose pyrophosphorylase from potato tuber: cDNA cloning and sequencing

We have isolated a cDNA encoding UDP-glucose pyrophosphorylase from a cDNA library of immature potato tuber using oligonucleotide probes synthesized on the basis of partial amino acid sequences of the enzyme. The cDNA clone contained a 1,758-base-pair insert including the complete message for UDP-glucose pyrophosphorylase with 1,431 base pairs. The amino acid sequence of the enzyme inferred from the nucleotide sequence consists of 477 amino acid residues. All the partial amino acid sequences determined protein-chemically [Nakano et al. (1989) J. Biochem. 106, 528-532] confirmed the primary structure of the enzyme. An N-terminal-blocked peptide was isolated from the proteolytic digest of the enzyme protein, and the blocking group was deduced to be an acetyl group by fast atom bombardment-mass spectrometry. On the basis of the predicted amino acid sequence (477 residues minus the N-terminal Met plus an acetyl group), the molecular weight of the enzyme monomer is calculated to be 51,783, which agrees well with the value determined by polyacrylamide gel electrophoresis. In the cDNA structure, the open-reading frame is preceded by a 125-base-pair noncoding region, which contains a sequence being homologous with the consensus sequence for plant genes, and is followed by a 174-base-pair noncoding sequence including a polyadenylation signal. Amino acid sequence comparisons revealed that the potato UDP-glucose pyrophosphorylase is homologous to the enzyme from slime mold, Dictyostelium discoideum, but not to ADP-glucose pyrophosphorylases from rice seed and Escherichia coli.