cDNA cloning and expression of rat and human protein geranylgeranyltransferase type-I

Protein geranylgeranyltransferase type-I (GGTase-I) transfers a geranylgeranyl group to the cysteine residue of candidate proteins containing a carboxyl-terminal CAAX (C, cysteine; A, aliphatic amino acid; X, any amino acid) motif in which the "X" residue is leucine. The enzyme is composed of a 48-kilodalton alpha subunit and a 43-kilodalton beta subunit. Peptides isolated from the alpha subunit of GGTase-I were shown to be identical with the alpha subunit of a related enzyme, protein farnesyltransferase. Overlapping cDNA clones containing the complete coding sequence for the beta subunit of GGTase-I were obtained from rat and human cDNA libraries. The cDNA clones from both species each predicted a protein of 377 amino acids with molecular masses of 42.4 kilodaltons (human) and 42.5 kilodaltons (rat). Amino acid sequence comparison suggests that the protein encoded by the Saccharomyces cerevisiae gene CDC43 is the yeast counterpart of the mammalian GGTase-I beta subunit. Co-expression of the GGTase-I beta subunit cDNA together with the alpha subunit of protein farnesyltransferase in Escherichia coli produced recombinant GGTase-I with electrophoretic and enzymatic properties indistinguishable from native GGTase-I.     

Molecular cloning and expression of cynomolgus monkey interleukin-2 cDNA by the recombinant baculovirus system

Mice inoculated at 5, 21 and 28 days of age with 10(6) or 10(7) Cryptosporidium parvum oocysts became infected but did not exhibit any clinical signs of disease. Specific IgA antibodies were detected in faecal extracts from all infected mice by an indirect immunofluorescent assay. These antibodies first appeared between 11 and 37 days post-infection (dpi) and persisted until the end of the experiment at 55 dpl. They appeared earlier in older mice than in newborn mice. Reduction and resolution of oocyst shedding was not directly related, however, to IgA antibody levels in infected mice. Reactive C. parvum antigens were identified by immunoblotting techniques using faecal and serum samples from infected mice. IgA copro-antibodies reacted specifically with two antigens of 26 and 33 kDa, which were also identified by IgG antibodies in mouse serum. The role of these antibodies in the resolution of infections and the subsequent protection against challenge is unknown.     

Molecular cloning and expression of a novel human cDNA containing CAG repeats

A novel human cDNA containing CAG repeats, designated B120, was cloned by PCR amplification. An approximately 300-bp 3' untranslated region in this cDNA was followed by a 3426-bp coding region containing the CAG repeats. A computer search failed to find any significant homology between this cDNA and previously reported genes. The number of CAG trinucleotide repeats appeared to vary from seven to 12 in analyses of genomic DNA from healthy volunteers. An approximately 8-kb band was detected in brain, skeletal muscle and thymus by Northern blot analysis. The deduced amino-acid sequence had a polyglutamine chain encoded by CAG repeats as well as glutamine- and tyrosine-rich repeats, which has also been reported for several RNA binding proteins. We immunized mice with recombinant gene product and established a monoclonal antibody to it. On Western immunoblotting, this antibody detected an approximately 120-kDa protein in human brain tissue. In addition, immunohistochemical staining showed that the cytoplasm of neural cells was stained with this antibody. These findings indicated that B120 is a novel cDNA with a CAG repeat length polymorphism and that its gene product is a cytoplasmic protein with a molecular mass of 120 kDa.     

Molecular cloning of a canine metallothionein cDNA

A canine metallothionein cDNA obtained from the liver of a cadmium-treated beagle was cloned and sequenced. Asn at position 4 conserved among all mammalian metallothionein-1 and metallothionein-2 is replaced by Asp in the canine metallothionein cDNA clone. Because the acidic amino acid doesn't exist at either position 10 or 11 in the deduced amino acid sequence, it is supposed that this cDNA is derived from canine metallothionein-1 mRNA. Northern blot analysis using the cDNA as a probe revealed the induction of the canine metallothionein mRNA expression in the liver and kidney of a cadmium-treated beagle. Thus, the canine metallothionein cDNA obtained in the present study should provide an useful tool for the molecular investigation of metallothionein in dog.     

Molecular cloning of rat cytokine synthesis inhibitory factor (IL-10) cDNA and expression in spleen and macrophages

Rat cytokine synthesis inhibitory factor (IL-10) was cloned. Like mouse and human IL-10 (mIL-10, hIL-10), rat IL-10 exhibits strong DNA and amino acid sequence homology to the open reading frame in the Epstein-Barr virus, BCRFI. The supernatant of COS-7 transfectants with rat IL-10 was found to inhibit the production of IFN-gamma by murine Th1 cells. A probe from the rat IL-10 was used in an RNase protection assay to demonstrate that IL-10 was expressed in LPS-stimulated rat spleen and FACS-purified OX-42-reactive peritoneal macrophages. The production of IL-10 by macrophages, as shown in the current study, suggests that IL-10 may have an autocrine function in inflammation.     

Molecular cloning of cDNA encoding porcine interleukin-15

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.     

Molecular cloning and functional expression of a human cDNA encoding translation initiation factor 6

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. In this paper, we devised a procedure for purifying eIF6 from rabbit reticulocyte lysates and immunochemically characterized the protein by using antibodies isolated from egg yolks of laying hens immunized with rabbit eIF6. By using these monospecific antibodies, a 1.096-kb human cDNA that encodes an eIF6 of 245 amino acids (calculated Mr 26,558) has been cloned and expressed in Escherichia coli. The purified recombinant human protein exhibits biochemical properties that are similar to eIF6 isolated from mammalian cell extracts. Database searches identified amino acid sequences from Saccharomyces cerevisiae, Drosophila, and the nematode Caenorhabditis elegans with significant identity to the deduced amino acid sequence of human eIF6, suggesting the presence of homologues of human eIF6 in these organisms.     

Molecular cloning and expression in Saccharomyces cerevisiae of tobacco NADPH-cytochrome P450 oxidoreductase cDNA

We obtained information on the full length tobacco NADPH-cytochrome P450 oxidoreductase (P450 reductase) by a combination of the cDNA clone pCTR1 and the genomic DNA clone pGTR1. The deduced primary structure consisting of 713 amino acid residues contained sequences corresponding to FMN, FAD, and NADPH-binding regions. Based on this information, we prepared the full-length cDNA pFTR of tobacco P450 reductase by RT-PCR and expressed it in the yeast Saccharomyces cerevisiae. The transformed yeast cells carrying pFTR produced the corresponding mRNA and protein, and had increased cytochrome c reductase activity in the microsomes. An in vitro reconstitution system of the yeast microsomal fractions expressed tobacco P450 reductase and rat P450 1A1 showed an increased 7-ethoxycoumarin O-deethylase activity. These results indicated that tobacco P450 reductase expressed in the yeast microsomes coupled with rat P450 1A1 resulting in an increased monooxygenase activity.     

Molecular cloning and functional expression of a cDNA for rat phospholipid hydroperoxide glutathione peroxidase: 3'-untranslated region of the gene is necessary for functional expression

A full-length cDNA clone for phospholipid hydroperoxide glutathione peroxidase (PHGPx) was isolated from a rat brain. The cDNA was 0.761 kb in length and encoded 170 amino acids, which included a TGA-encoded selenocysteine at residue 46. The protein has a calculated molecular mass of 19,473 Da. We succeeded in the transient functional expression of a full-length cDNA for PHGPx, which includes the 3'-UTR, in COS-7 cells at the first attempt. Deletion of the 3'-UTR prevented the expression of the PHGPx activity and the incorporation of [75Se]selenious acid into the monomeric 19.7 kDa PHGPx protein. Thus, the 3'-UTR of the cDNA for PHGPx was required for the functional expression of PHGPx. Northern blot analysis demonstrated that the mRNA for PHGPx was widely expressed in normal rat tissues, especially in the testis. The mRNA levels of PHGPx in the cultured cells such as hepatomas, neuronal cells, nephroblastoma, and mammary myo-epithelial cells were higher than those of the tissues. The ratio of PHGPx to cytosolic glutathione peroxidase (cGPx) was significantly high in the testis and relatively high in the cultured cells. The mRNA levels of PHGPx in tissues were lower than those of cGPx.     

Molecular cloning and functional expression of rat liver glutathione-dependent dehydroascorbate reductase

We have isolated a cDNA clone for a novel glutathione-dependent dehydroascorbate reductase from a rat liver cDNA library in lambdagt11 by immunoscreening. The authenticity of the clone was confirmed as follows: first, the antibody that had been purified through affinity for the protein expressed by the cloned lambdagt11 phage recognized only the enzyme in a crude extract from rat liver; and second, two internal amino acid sequences of purified enzyme were identified in the protein sequence predicted from the cDNA. The predicted protein consists of 213 amino acids with a molecular weight of 24,929, which is smaller by approximately 3,000 than the value obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This discrepancy of the molecular weight was explained by post-translational modification because the recombinant protein expressed by a mammalian system (Chinese hamster ovary cells) was of the same size as rat liver enzyme but larger than the protein expressed by a bacterial system (Escherichia coli). Chinese hamster ovary cells, originally devoid of glutathione-dependent dehydroascorbate reductase activity, was made to elicit the enzyme activity (1.5 nmol/min/mg of cytosolic protein) by expression of the recombinant protein. Additionally, the cells expressing the enzyme were found to accumulate 1.7 times as much ascorbate as the parental cells after incubation with dehydroascorbate. This result points to the importance of the dehydroascorbic acid reductase in maintaining a high concentration of ascorbate in the cell.     

Molecular cloning of human class IV alcohol dehydrogenase cDNA

A cDNA encoding a new type of alcohol dehydrogenase was cloned from a human stomach cDNA library. PCR amplification of 5'-stretch human stomach lambda gt11 library, using degenerate inosine-containing oligonucleotide probes compatible with peptide sequences of human sigma-ADH, resulted in a single product. Subsequently, internal non-degenerate primers were constructed according to the sequences occurring in the product. By PCR with combinations of these new primers and lambda gt11 forward and reverse primers, fragments of the cDNA containing its 5' and 3' ends were amplified. The full length cDNA sequence has 1125 nucleotides with a 72% similarity to those of human class I ADH. The polypeptide sequence, predicted from the cDNA, corresponds to 373 amino acids with a high degree of similarity (96%) to fragments of sigma-ADH previously reported. Northern hybridization analysis with the specific probe for the mRNA of this protein showed that it is expressed in the human stomach but not in the liver. These data indicate that the cDNA we cloned is that of human class IV ADH.     

Molecular cloning and expression of the functional rat C5a receptor

The C5a receptor belongs to the superfamily of G-protein coupled receptors with seven transmembrane segments. In this study we report on the cloning of the rat C5a receptor (ratC5aR). We used a hybridization probe produced by PCR utilizing degenerate primers which corresponded to conserved parts of the human, canine and murine C5a receptor nucleotide sequences and to the published partial amino acid sequence of the rat C5a receptor to screen a rat macrophage cDNA library. We found two overlapping clones containing an open reading frame of 1056 bp, a 3'untranslated region of 683 bp and a 5'untranslated region of 27 bp. The overall nucleotide acid sequence identity, compared to the murine, human and canine C5a receptor sequences, was 85.8, 70.5 and 68.9%, respectively. The greatest diversity exists in the putative extracellular domains, especially in the aminoterminal domain which is assumed to be involved in ligand binding. An N-glycosylation site is present within the N-terminal sequence at residue 6. One of the cDNA containing the 5'untranslated region, the coding sequence and part of the 3'untranslated region was cloned into an eucaryotic expression vector and stably transfected into the rat basophilic leukemia cell line RBL-2H3. Expression of the rat C5a receptor on the surface of these cells could be demonstrated by flow cytometric analysis using FITC-labeled recombinant rat C5a (rrC5a). By measuring the release of calcium from intracellular stores after stimulation with rrC5a it could further be shown that the receptor is functionally coupled. Receptor binding assays showed that rrC5a specifically binds to the ratC5aR with a KD of 0.91 +/- 0.36 and to the human C5a receptor (huC5aR) with a KD of 7.19 +/- 1.56. The determined KD for binding of human C5a (huC5a) to the huCSaR was 2.16 +/- 0.65. No binding of huC5a to the ratC5aR could be observed although high concentrations of this ligand (> 60 nM) promoted chemotaxis of RBL cells transfected with the huC5aR.     

Molecular cloning and functional analysis of cDNA encoding a rat leukemia inhibitory factor: towards generation of pluripotent rat embryonic stem cells

Embryonic stem (ES) cells are pluripotent cell lines established directly from the early embryo. Maintenance of the stem-cell phenotype of ES cells in vitro requires the presence of a feeder layer of fibroblasts or of a soluble factor, differentiation inhibitory activity (DIA) such as leukemia inhibitory factor (LIF). Here we report the cloning of complete rat LIF cDNA and its nucleotide sequence so as to facilitate studies of rat ES cell technologies on tumor biology. The nucleotide sequence of the rat LIF cDNA indicated that the rat LIF has 91% amino acid sequence identity with murine LIF. The cloned rat LIF cDNA has a putative biological activity as a differentiation-inducing factor on the murine myeloid leukemia cell line M1 cells. Culture supernatant of the rat LIF cDNA-transduced rat fibroblast cell line could maintain the stem-cell phenotype of rat ES cells which showed alkaline phosphatase activity, and this effect was much stronger than that by murine LIF. The availability of rat LIF which shows DIA will assist the in vitro analysis of rat ES cells, and culture of these cells is a route for the generation of gene targeting in rat.     

Molecular cloning and sequencing of the cDNA coding for a calcium-binding protein regucalcin from rat liver

The 3-years-old patient presents bilateral exophthalmia and papillar stasis at both eyes. The cranial examination shows the frontal boss at the level of the anterior fontanelle and the hypoplasia of the inferior facial floor. The cranial radiography shows digital impressions on the entire projection surface of the cap. The child also presents mental retardation. The affection integrates in the category of craniofacial dysostosis described by Crouzon, maladies which are transmitted autosomal with great penetrance.     

水稻PHD锌指蛋白cDNA的克隆及其表达分析

利用cDNA-AFLP分析籼稻明恢86应答稻纵卷叶螟取食基因差异表达,发现1个与植物同源结构域(PHD)锌指蛋白高度同源的TDF,分离获得该TDF对应的粳稻全长cDNA.该全长cDNA与籼稻、玉米、蓖麻、葡萄、拟南芥和大豆等作物PHD锌指蛋白推定氨基酸序列的同源性分别为98.43%、86.01%、54.58%57.02%、57.51%和55.88%.荧光定量PCR研究表明,日本晴(粳稻)叶片中该基因的表达也受稻纵卷叶螟取食的诱导,推测该基因与籼稻和粳稻应答稻纵卷叶螟取食密切相关.