Molecular cloning of the cDNA encoding an immunodominant antigen of Dirofilaria immitis.

An immunodominant antigen of Dirofilaria immitis was studied using recombinant DNA techniques. The mRNA from D. immitis adult female worms was translated in vitro and a major 34 kDa antigenic polypeptide product was demonstrated by immunoprecipitation. cDNA was synthesized from mRNA and a lambda gt11 expression library was constructed and immunoscreened with dirofilariasis positive serum. A positive clone containing a nearly full length cDNA was isolated. The cDNA was 2415 bp in length and consisted of a single open reading frame followed by a long 3' non-coding region of 1446 bp. The open reading frame of 969 bp encoded a polypeptide of 322 amino acids with a molecular weight of 34,400. A cDNA fusion protein synthesized by bacteria (Escherichia coli JM109) using the expression vector pGEMEX-1 was identified as an immunodominant antigen by absorption experiments and had no cross-reactivity with sera from patients with other filarial species.     

Molecular cloning of cDNA for antimycin A-inducible mRNA and its role in cyanide-resistant respiration in Hansenula anomala

A cDNA for mRNA induced by antimycin A in Hansenula anomala was cloned. The mRNA for the cDNA was expressed in the yeast under the conditions expressing the cyanide-resistant respiration activity. The nucleotide sequence revealed a long open reading frame of 342 codons encoding a protein with a molecular weight of 40,282 in the cDNA. An antibody recognizing the protein encoded by the open reading frame was produced. Immunoblotting of H. anomala proteins with this antibody showed that a 36 kDa protein localized in mitochondria was a mature form of the protein encoded by the cDNA. It is suggested that the cloned cDNA encodes a protein involved in the cyanide-resistant respiratory pathway.     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

甘蓝夜蛾几丁质酶基因cDNA序列的克隆与序列分析

昆虫几丁质酶在害虫生物防治中具有很大的发展潜力。以甘蓝夜蛾Mamestra brassicae L.预蛹期幼虫整个虫体为材料提取总RNA,利用RT-PCR和cDNA末端快速扩增技术(RACE),扩增得到其几丁质酶的cDNA序列。该序列含有2826个碱基,包括1个1689个碱基的开放阅读框,预测编码1个含562个氨基酸的多肽,分子量约为62.6kDa,等电点为5.30。推导得到的氨基酸序列含有2个N-位糖基化位点,22个O-位糖基化位点,氨基酸序列与其他昆虫,尤其是鳞翅目昆虫的几丁质酶高度同源。获得的甘蓝夜蛾几丁质酶基因cDNA序列已经登录GenBank并获得登录号FJ436415。     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.     

Molecular cloning and functional analysis of a human cDNA encoding an Escherichia coli AlkB homolog, a protein involved in DNA alkylation damage repair.

The Escherichia coli AlkB protein is involved in protecting cells against mutation and cell death induced specifically by SN2-type alkylating agents such as methyl methanesulfonate (MMS). A human cDNA encoding a polypeptide homologous to E.coli AlkB was discovered by searching a database of expressed sequence tags (ESTs) derived from high throughput cDNA sequencing. The full-length human AlkB homolog (hABH) cDNA clone contains a 924 bp open reading frame encoding a 34 kDa protein which is 52% similar and 23% identical to E.coli AlkB. The hABH gene, which maps to chromosome 14q24, was ubiquitously expressed in 16 human tissues examined. When hABH was expressed in E.coli alkB mutant cells partial rescue of the cells from MMS-induced cell death occurred. Under the conditions used expression of hABH in skin fibroblasts was not regulated by treatment with MMS. Our findings show that the AlkB protein is structurally and functionally conserved from bacteria to human, but its regulation may have diverged during evolution.     

The cloning and characterization of Tetrahymena pyriformis translation elongation factor 1b alpha and gamma subunits

The multi-subunit eukaryotic translation elongation factor 1 (eEF1) consists of two functionally distinct parts: G-protein eEF1A and guanine nucleotide exchange factor eEF1B. Here, we report on the cloning of cDNAs of both the alpha and gamma subunits of the eEF1B from the ciliated protozoan Tetrahymena pyriformis. The open reading frame of the eEF1Bgamma cDNA encodes a 399-amino acid long polypeptide with a calculated molecular mass of 45.2 kDa. The eEF1Balpha cDNA contains an open reading frame encoding a polypeptide of 228 amino acids. The calculated molecular mass of this protein is 25.2 kDa. The overall deduced amino acid sequences of eEF1Balpha and eEF1Bgamma show a considerable homology with the families of alpha and gamma proteins from other eukaryotic organisms. We demonstrated that eEF1Bgamma is an RNA-binding protein which is able to bind to different RNAs.     

Cloning and expression of solanidine UDP-glucose glucosyltransferase from potato

A cDNA encoding solanidine glucosyltransferase (SGT) was isolated from potato. The cDNA was selected from a yeast expression library using a positive selection based on the higher toxicity of steroidal alkaloid aglycons relative to their associated glycosylated forms. The cDNA contained an open reading frame encoding a 56 kDa polypeptide with regions of similarity to previously characterized UDP-glucosyltransferases. The enzyme activity and reaction products of recombinant SGT in yeast were consistent with those observed for the endogenous enzyme from potato. SGT mRNA and protein accumulated in tubers in response to wounding. The time course for SGT mRNA accumulation paralleled that of 3-hydroxy-3-methylglutaryl-coenzymeA isoform 1 ( hmg1 ) mRNA. Steady-state SGT mRNA levels also increased transiently upon wounding of leaves.     

Isolation and sequence analysis of a cDNA encoding the ATP/ADP translocator of Zea mays L.

A cDNA complementary to the mRNA for the ATP/ADP translocator of maize (Zea mays L.) has been identified by virtue of hybridisation with the homologous gene from yeast. The cloned cDNA has been shown by DNA sequence analysis to contain an open reading frame of 954bp., which encodes a polypeptide of molecular weight 40,519. This polypeptide exhibits a high degree of homology to the translocator polypeptides of beef heart and Neurospora crassa mitochondria.     

Cloning of cDNA encoding a soybean allergen, Gly m Bd 28K

A cDNA clone encoding a soybean allergen, Gly m Bd 28K, has been isolated. The clone has a 1567-bp cDNA insert with a 1419-bp open reading frame and a 148-bp 3'-untranslated region, followed by a polyadenylation tail. The open reading frame was shown to encode a polypeptide composed of 473 amino acids. The chemically determined amino acid sequences of the peptides obtained from the allergen, including its N-terminal peptide, were shown to be contained in the N-terminal region of the amino acid sequence deduced from the cDNA, showing that the first half of the cDNA encodes the allergen with a preceding segment of 21 amino acids. The peptide fragment including the allergen was expressed as a fusion protein with glutathione S-transferase in Escherichia coli and immunoblotted with the sera of soybean-sensitive patients and the monoclonal antibody against the allergen. Furthermore, homology analyses demonstrate that the polypeptide for the cDNA exhibits high homology with the MP27/MP32 proteins in pumpkin seeds and the carrot globulin-like protein. This finding suggests that the polypeptide may consist of a 21-amino acid segment as a part of the signal peptide and the proprotein, which may be converted to two mature proteins, Gly m Bd 28K and a 23-kDa protein, during the development of soybean cotyledons.     

Purification, characterization, and cDNA cloning of an AU-rich element RNA-binding protein, AUF1.

The degradation of some proto-oncogene and lymphokine mRNAs is controlled in part by an AU-rich element (ARE) in the 3' untranslated region. It was shown previously (G. Brewer, Mol. Cell. Biol. 11:2460-2466, 1991) that two polypeptides (37 and 40 kDa) copurified with fractions of a 130,000 x g postribosomal supernatant (S130) from K562 cells that selectively accelerated degradation of c-myc mRNA in a cell-free decay system. These polypeptides bound specifically to the c-myc and granulocyte-macrophage colony-stimulating factor 3' UTRs, suggesting they are in part responsible for selective mRNA degradation. In the present work, we have purified the RNA-binding component of this mRNA degradation activity, which we refer to as AUF1. Using antisera specific for these polypeptides, we demonstrate that the 37- and 40-kDa polypeptides are immunologically cross-reactive and that both polypeptides are phosphorylated and can be found in a complex(s) with other polypeptides. Immunologically related polypeptides are found in both the nucleus and the cytoplasm. The antibodies were also used to clone a cDNA for the 37-kDa polypeptide. This cDNA contains an open reading frame predicted to produce a protein with several features, including two RNA recognition motifs and domains that potentially mediate protein-protein interactions. These results provide further support for a role of this protein in mediating ARE-directed mRNA degradation.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

一个新的忽地笑O-甲基转移酶基因的克隆与原核表达

采用RACE技术克隆得到一个新的忽地笑O-甲基转移酶（OMT）基因,命名为LaOMT。LaOMT的cDNA序列为1379 bp,开放阅读框1077 bp,预测编码蛋白包含358个氨基酸。序列比对分析发现, LaOMT编码的蛋白序列具有依赖于S-腺苷-L-甲硫氨酸（SAM）的高度保守的结构域,与葡萄、大豆等其他植物OMTs蛋白的相似性为50%左右。将LaOMT基因连接到原核表达载体pET28a上,转化大肠杆菌Rosetta （DE3）的SDS-PAGE电泳结果显示,重组蛋白受异丙基硫代半乳糖苷（IPTG）诱导表达,分子量大小约为45 kDa,与已报导的其他植物的OMTs蛋白大小基本一致。此外,半定量RT-PCR分析结果表明, LaOMT在忽地笑的叶、鳞茎、根和花中均有表达,其中花中的表达量最低。     

Molecular cloning of the human ATP-binding cassette transporter 1 (hABC1): evidence for sterol-dependent regulation in macrophages

We have cloned the full-length cDNA for the human ATP binding cassette transporter 1 (hABC1). The 6603-bp open reading frame encodes a polypeptide of 2201 amino acids resulting in a deduced molecular weight of 220 kDa. The hABC1 cDNA is highly homologous (62%) to the human rim ABC transporter (ABCR). hABC1 is expressed in a variety of human tissues with highest expression levels found in placenta, liver, lung, adrenal glands, and fetal tissues. We demonstrate that the hABC1 expression is induced during differentiation of human monocytes into macrophages in vitro. In macrophages, both the hABC1 mRNA and protein expression are upregulated in the presence of acetylated low-density lipoprotein (AcLDL). The AcLDL-induced increase in hABC1 expression is reversed by cholesterol depletion mediated by the addition of high-density lipoprotein (HDL3). Our data, demonstrating sterol-dependent regulation of hABC1 in human monocytes/macrophages, suggest a novel role for this transporter molecule in membrane lipid transport.     

Cloning of cDNAs encoding the 160 kDa subunit of the bovine cleavage and polyadenylation specificity factor.

3'-processing of mRNA precursors depends on several protein factors. One of them, cleavage and polyadenylation specificity factor (CPSF) is required for the cleavage of the mRNA precursor or as well as for the tail elongation reaction. We have obtained complementary DNA encoding the 160 kDa subunit, which had previously been shown to interact with the AAUAAA polyadenylation signal. The cDNAs code for an open reading frame of 1444 amino acids. The translated protein has a calculated molecular weight of 161 kDa and a predicted pl of 6.2. Polyclonal antibodies raised against a bacterially expressed fragment of the cDNA recognise 160 kDa subunit of purified calf thymus CPSF. The sequence contains a possible nuclear localisation signal but none of the known RNA binding motifs. It does, however, show sequence similarities to a UV-damaged DNA binding protein (UVdDb).