Comparison of acetylcholinesterase genes from cattle ticks

We describe the isolation and characterisation of two putatively new acetylcholinesterase genes from the African cattle ticks Boophilus decoloratus and Rhipicephalus appendiculatus. The nucleotide sequences of these genes had 93% homology to each other and 95% and 91% identity, respectively, to the acetylcholinesterase gene from an Australian strain of another cattle tick, Boophilus microplus. Translation of the nucleotide sequences revealed putative amino acids that are essential for acetylcholinesterase activity: the active site serine, and the histidine and glutamate residues that associate with this serine to form the catalytic triad. All known acetylcholinesterases have three sets of cysteines that form disulfide bonds; however, the acetylcholinesterase genes of these three species of ticks encode only two sets of cysteines. Acetylcholinesterases of B. microplus from South Africa, Zimbabwe, Kenya and Mexico had 98-99% identity with acetylcholinesterase from B. microplus from Australia, whereas acetylcholinesterase from B. microplus from Indonesia was identical to that from Australia. Preliminary phylogenetic analyses surprisingly indicate that the acetylcholinesterases of ticks are closer phylogenetically to acetylcholinesterases of vertebrates than they are to those of other arthropods.     

cDNA sequence and expression of a phosphoenolpyruvate carboxylase gene from soybean

A full-length cDNA encoding a subunit of phosphoenolpyruvate carboxylase (PEPC) was isolated from a developing seed expression library of the C₃ plant Glycine max. The corresponding mRNA is present at similar levels in leaf, stem, root and developing seed. Two potential start codons exist, and the activity of protein initiated from the first such codon could be subject to regulation by protein kinase. Sequence comparison shows a similar upstream start codon in the case of the Ppc2 gene from Mesembryanthemum crystallinum, previously assumed to lack the sequences necessary for phosphorylation. The soybean encoded protein tends to resemble other C₃-type PEPC proteins more closely than those implicated in C₄ or crassulacean acid metabolism.     

Taenia solium cDNA sequence encoding a putative immunodiagnostic antigen for human cysticercosis

A T. solium metacestode cDNA library was prepared and antibody screened to obtain recombinant antigens, which could be used for the neurocysticercosis diagnosis. The F18 clone was selected and sequenced, and the full length cDNA characterised as well as the genomic structure from the gene. F18 is a single copy gene that spans approximately 6.1 kb and contains five exons and four introns. The F18 cDNA has a 690-nucleotide open reading frame that encodes a putative polypeptide of 229 amino acids with a predicted molecular mass of 26.06 x 10(3) M(r). The F18 recombinant protein was obtained and purified by affinity chromatography using pGEX system (G-F18) and pQE system (H-F18). The purified G-F18 fusion protein showed the best results when it was used in ELISA with sera from neurocysticercosis patients.     

Cyclic GMP phosphodiesterase from cattle retina. Amino acid sequence of the gamma-subunit and nucleotide sequence of the corresponding cDNA

The primary structure of the gamma-subunit of cyclic GMP phosphodiesterase was determined by parallel analysis of the amino acid sequence of the protein and nucleotide sequence of the corresponding cDNA. The enzyme gamma-subunit contains 87 amino acid residues, its N-terminal amino group being acetylated.     

家蝇泛素编码区 cDNA 序列的克隆及在原核细胞中的表达

泛素-蛋白酶体途径(ubiquitin-proteasome pathway)是具有高度选择性的蛋白质降解途径,该途径对细胞内蛋白的选择性降解起着重要作用。本研究根据 GenBank 已登录的真核生物泛素（ubiquitin）编码框的氨基酸序列,设计一对简并引物,RT-PCR 克隆了家蝇 Musca domestica 泛素基因的编码区 cDNA 序列,并进行了测序。序列分析表明,该编码区的长度为 228 bp,编码 76 个氨基酸,命名为 Mdubi ,GenBank 登录号为 DQ115796。同源性比较发现,Mdubi 氨基酸序列与其他真核生物泛素编码框同源性可达 94% 以上。RT-PCR 检测表明,Mdubi 在家蝇不同组织中均高效表达,且不受大肠杆菌 Escherichia coli 刺激的影响,是遍在性表达。为进一步研究 Mdubi 的结构和功能,将 Mdubi 克隆到原核表达载体 pQE30 上,构建重组质粒 pQE30-UBI,转化大肠杆菌 M15 感受态细胞,在 IPTG 诱导下进行了高效表达,SDS-PAGE 检测表明 Mdubi 在大肠杆菌中可表达相对分子质量为 9.6 kD 的可溶性融合蛋白;Western blot 分析表明表达产物能与 Ni-NTA 鏊合物特异性的结合,表明表达的 Mdubi 为 N 端带有 6His 标签的融合蛋白。利用 Ni²⁺-NTA 亲和柱一步纯化了 Mdubi,以该融合蛋白免疫新西兰大白兔制备了抗 Mdubi 血清。本研究成功克隆了家蝇泛素的编码序列,并在原核细胞中得到了表达,为进一步研究泛素在家蝇体内的作用机制奠定了基础。     

Cloning and expression of a putative transferrin cDNA of the spruce budworm, Choristoneura fumiferana

A spruce budworm (Choristoneura fumiferana) transferrin cDNA (CfTf) was isolated and cloned from a cDNA library that was constructed using mRNA from fifth to sixth instar larvae. CfTf cDNA encoded a predicted protein of 681 amino acids with a molecular mass of approximately 76 kDa. CfTf shared 72% and 74% identities at the amino acid level with transferrins of Manduca sexta and Bombyx mori, respectively. Like other transferrins, CfTf retains most of the N-terminal, iron-binding amino acid residues. Northern blot analyses indicated that CfTf mRNA was present at high levels after ecdysis, but that the expression level was low prior to ecdysis at the fourth-sixth instar stages. The highest level of CfTf expression was detected in the fat body. Relatively low levels of expression were detected in the epidermis and no expression was found in the midgut. Expression of CfTf mRNA could be induced by bacteria but not fungi. Expression of CfTf mRNA was suppressed by iron load.     

Confirming single nucleotide polymorphisms from expressed sequence tag datasets derived from three cattle cDNA libraries

Using the Phred/Phrap/Polyphred/Consed pipeline established in the National Livestock Research Institute of Korea, we predicted candidate coding single nucleotide polymorphisms (cSNPs) from 7,600 expressed sequence tags (ESTs) derived from three cDNA libraries (liver, M. longissimus dorsi, and intermuscular fat) of Hanwoo (Korean native cattle) steers. From the 7,600 ESTs, 829 contigs comprising more than two EST reads were assembled using the Phrap assembler. Based on the contig analysis, 201 candidate cSNPs were identified in 129 contigs, in which transitions (69%) outnumbered transversions (31%). To verify whether the predicted cSNPs are real, 17 SNPs involved in lipid and energy metabolism were selected from the ESTs. Twelve of these were confirmed to be real while five were identified as artifacts, possibly due to expressed sequence tag sequence error. Further analysis of the 12 verified cSNPs was performed using the program BLASTX. Five were identified as nonsynonymous cSNPs, five were synonymous cSNPs, and two SNPs were located in 3'-UTRs. Our data indicated that a relatively high SNP prediction rate (71%) from a large EST database could produce abundant cSNPs rapidly, which can be used as valuable genetic markers in cattle.     

cDNA sequence and expression of a cold-responsive gene in Citrus unshiu

A cDNA clone encoding a protein (CuCOR19), the sequence of which is similar to Poncirus COR19, of the dehydrin family was isolated from the epicarp of Citrus unshiu. The molecular mass of the predicted protein was 18,980 daltons. CuCOR19 was highly hydrophilic and contained three repeating elements including Lys-rich motifs. The gene expression in leaves increased by cold stress.     

Revised sequence and expression of cyclin B cDNA from the starfish Asterina pectinifera.

Cyclin B cDNA was cloned from the ovary of the starfish Asterina pectinifera and analyzed by RT-PCR and 3'- and 5'-RACE techniques. The cDNA consists of a 0.13-kb upstream untranslated region, a 1.22-kb coding region, and a 0.86-kb downstream untranslated region. The open reading frame encoded a polypeptide of 404 amino acid residues with a calculated molecular weight of 45,692. All the characteristic sequences, such as destruction and cyclin boxes, cyclin B motif, and cytoplasmic retention and nuclear export signals, were found in the newly cloned cyclin B cDNA. The deduced amino acid sequence of the cyclin B cDNA was highly homologous in the middle and carboxy terminal regions to that from mature eggs of the same organism, but quite different in the amino terminal region. Evidence was obtained which suggested that this cyclin B is expressed in immature and maturing oocytes and is the same as that cloned from mature eggs.     

cDNA of cattle alpha S1-casein: cloning and nucleotide sequence

The nucleotide sequence of recombinant plasmids representing a full-size cDNA of cow alpha s1-casein was investigated. The corresponding mRNA consists of 1133 nucleotides except for poly(A) and includes 642 nucleotides of the coding region, 63 nucleotides of 5'- and 428 nucleotides of the 3'-noncoding regions. A comparative analysis of nucleotide sequences of cow alpha s1-casein and guinea pig B-casein showed that the homology in the 5'-nontranslatable region is 90.5%, that of a precasein single peptide is 82.22%, while that of the major polypeptide in the coding region is 64% without taking into account the blank spaces. The homology is higher in the 3'-noncoding region than in the coding region and makes up to 72%. The data obtained testify to the high degree of conservatism of sequences in casein mRNA noncoding regions as well as to functional and regulatory role of these sequences in gene expression of caseins.