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.     

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.     

cDNA cloning and expression analysis of the murine ribonuclease L inhibitor

This study was designed to test the hypothesis that the immune changes seen during in vivo whole body hyperthermia are mediated by elevations in the plasma concentrations of either catecholamines, growth hormone or beta-endorphins. Eight healthy volunteers were immersed in a hot water bath (WI; water temperature 39.5 degrees C) for 2 h during which their rectal temperature rose to 39.5 degrees C. In a single blind, randomized, cross-over study the stress hormone effects were blocked one at a time by administration of propranolol, somatostatin or naloxone; the results were compared to those obtained during saline infusion (control). Blood samples were collected before, at the end of 2 h of WI (body temperature 39.5 degrees C), and 2 h later. Hormone blockade did not abolish the hyperthermia-induced recruitment of natural killer (NK) cells to the blood, and no influence was observed on the percentages or concentrations of any other subpopulations of blood mononuclear cells, except that the number of cluster designation (CD)3+ cells slightly increased after hyperthermia only in the propranolol experiment. Furthermore, the NK cell activity, both unstimulated and interferon-alpha or interleukin-2 stimulated, did not differ from the control situation. It is of interest, however, that somatostatin partly abolished the hyperthermia induced increase in the neutrophil number. Based on these data and previous results showing that growth hormone infusion increases the concentration of neutrophils in the blood, it is suggested that growth hormone is at least partly responsible for hyperthermia induced neutrocytosis.     

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.     

Purification, cDNA cloning and heterologous expression of human phosphomannose isomerase

Phosphomannose isomerase catalyses the interconversion of fructose-6-P and mannose-6-P and has a critical role in the supply of D-mannose derivatives required for many eukaryotic glycosylation reactions. Three classes of enzymes possessing phosphomannose-isomerase activity have been identified in bacteria and lower eukaryotes. We have purified human phosphomannose isomerase to homogeneity from placental tissue. Protein sequence information obtained from internal fragments of the protein was used to design degenerate oligonucleotides which were used to amplify a fragment of a human phosphomannose-isomerase cDNA. A full-length cDNA was isolated from a human testes lambda gt11 library using this fragment as a probe. The cDNA encoded a protein with significant sequence identity to fungal and some bacterial phosphomannose isomerases but was unrelated to those from other bacteria. Based on amino acid sequence identity we propose a classification system for enzymes with phosphomannose-isomerase activity. The cDNA, under the control of the GAL1 promoter, was expressed in a Saccharomyces cerevisiae strain from which the native gene encoding phosphomannose isomerase had been deleted. The human enzyme was found to be able to functionally substitute for the yeast enzyme. Phosphomannose-isomerase mRNA was found in all human tissues tested but was more highly expressed in heart, brain and skeletal muscle. The cDNA was expressed in Escherichia coli permitting the isolation of pure recombinant protein which will be used for kinetic and structural studies.     

cDNA cloning and expression of a new form of human aryl sulfotransferase

Twenty-eight patients (with 30 primary and 8 revision total hip arthroplasties) completed a standardized questionnaire containing the Western Ontario and McMaster Universities (WOMAC) osteoarthritis index and Harris hip score questions prior to an office visit a minimum of 1 year after surgery. The range of hip motion measured by an orthopaedic surgeon was compared with the responses to questions on stiffness and function as well as with global scores in the WOMAC osteoarthritis index. Patient responses to the questions asking if they could cut their toenails on the operated side and the Harris hip score question asking if they could put on socks and tie a shoe correlated significantly with postoperative hip motion (P < .005). The WOMAC global pain and stiffness scores did not correlate with range of motion. The WOMAC physical function score correlated significantly only with hip flexion (P < .05). Of the WOMAC physical function questions, difficulty bending to pick an object off the floor (P < .05) and getting on and off the toilet (P < .05) correlated with the sum of the range of motion in all planes and weighted Harris hip score range of motion calculation. These data suggest that the points allocated in the Harris hip score for range of motion can be estimated reasonably accurately from questionnaire or phone response to a series of questions on a standardized questionnaire. The question on ability to cut toenails or the Harris hip score question regarding ability to put on socks and tie a shoe correlated with the most individual planes of motion, but several WOMAC physical function questions also correlated with total and weighted range of motion calculations.     

Purification, cDNA cloning and expression of human NG,NG-dimethylarginine dimethylaminohydrolase

cDNA encoding N(G),N(G)-dimethylarginine dimethylaminohydrolase from rat kidney had been cloned [Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N. & Ogawa, T. (1997) Biochim. Biophys. Acta 1337, 6-10]. The enzyme hydrolyzes N(G),N(G)-dimethyl-L-arginine and N(G)-monomethyl-L-arginine, which are known as endogenous inhibitors for the nitric oxide-generating system. In the present study, human N(G),N(G)-dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized. The cDNA clone encoding human N(G),N(G)-dimethylarginine dimethylaminohydrolase was isolated from a human kidney lambda gt10 library using a probe prepared from a plasmid containing the entire coding region of rat N(G),N(G)-dimethylarginine dimethylaminohydrolase. Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31,121 Da. The deduced amino acid sequence exhibits 93% identity with that of rat. The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme.     

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.     

cDNA cloning and expression of a soluble epoxide hydrolase from human liver

We report the cloning and expression of a cDNA that encodes a soluble epoxide hydrolase from human liver. The 2101-base clone predicts a 554-residue protein (M(r) 62,640) with an apparently imperfect peroxisomal targeting signal of Ser-Lys-Met at the carboxy terminus. The cDNA was expressed in the baculovirus system in the Spodoptera frugiperda 21 cell line. The recombinant protein was similar to soluble epoxide hydrolase isolated from human liver in terms of molecular weight, hydrolytic activity, inhibition, and immunoreactivity.     

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.     

Chicken keratin-19: cloning of cDNA and analysis of expression in the chicken embryonic gut

From many recent studies, it has been argued that keratins (cytokeratins) play important roles in the morphogenesis and differentiation of organ development. To learn the role of keratin in digestive tract development, a cDNA of the chicken homolog of keratin-19 (GK-19) was cloned and its expression pattern was analyzed in the digestive tract of chicken embryos. The GK-19 full-length sequence was approximately 1.6 kb and showed more than 80% similarity to human and mouse keratin-19. The result of in situ hybridization with the proventriculus (glandular stomach) of different developmental stages showed that GK-19 expression disappeared specifically in the glandular epithelium from day 6 to day 9 of incubation. Furthermore, GK-19 was localized in the notochord, floor plate, anterior lobe of the pituitary gland and mesonephros. These results suggest the possibility that GK-19 may have multiple roles in organogenesis during embryogenesis.     

cDNA cloning and characterization of a new human microsomal NAD+-dependent dehydrogenase that oxidizes all-trans-retinol and 3alpha-hydroxysteroids

We report the cDNA sequence and catalytic properties of a new member of the short chain dehydrogenase/reductase superfamily. The 1134-base pair cDNA isolated from the human liver cDNA library encodes a 317-amino acid protein, retinol dehydrogenase 4 (RoDH-4), which exhibits the strongest similarity with rat all-trans-retinol dehydrogenases RoDH-1, RoDH-2, and RoDH-3, and mouse cis-retinol/androgen dehydrogenase (相似文献   

Molecular cloning of a full-length cDNA for human type 3 adenylyl cyclase and its expression in human islets

The GK (Goto-Kakizaki) rat is a lean model of type 2 diabetes in which the diabetic state was spontaneously induced. We recently demonstrated the presence in GK rats of two functional point mutations in the promoter region of the type 3 adenylyl cyclase (AC3) gene that resulted in overexpression of AC3 mRNA associated with increased cAMP generation. The AC3 gene promoter mutations are the first molecular changes to be described in any specific gene in the GK rat. Here we report cloning of a full-length cDNA encoding human AC3 from a human fetal brain cDNA library using a PCR-based screening method. This 4142-bp cDNA predicts an open reading frame encoding 1144 amino acids containing putative 12 transmembrane-spanning domains which are typically found in other mammalian AC isoforms. Comparison of the translated amino acid sequence of the AC3 gene between human and rat shows 95% homology. Using RT-PCR, clear AC3 expression was detected in isolated human islets as well as a cDNA panel containing templates from eight different tissues (brain, heart, kidney, liver, lung, pancreas, placenta, and skeletal muscle). This wide distribution of AC3 expression may involve a number of physiological and pathophysiological metabolic processes.     

cDNA cloning and expression of a novel serine protease, TLSP

A cDNA for a putative novel serine protease, TLSP, was cloned from human hippocampus cDNA with polymerase chain reaction based strategies. The putative amino acid sequence of TLSP is similar to the trypsin-type serine proteases. TLSP mRNA is expressed in keratinocytes. Overexpressed TLSP protein in neuro2a cells was detected in culture medium.     

cDNA cloning and gene expression of phenylalanine ammonia-lyase in Lithospermum erythrorhizon

Two cDNA clones (LEPAL-1 and LEPAL-2) encoding phenylalanine ammonia-lyase were isolated from cell suspension cultures of Lithospermum erythrorhizon. Northern kinetic studies showed that LEPAL-1 mRNA contents markedly increased one day after inoculation of the cells into fresh medium, then decreased to the steady-state level. The course of mRNA accumulation paralleled that of PAL enzyme activity. The rapid induction of PAL activity seems to reflect the induction of dihydroechinofuran biosynthesis, while shikonin was produced at the steady-state level of PAL activity. The course of LEPAL-2 mRNA accumulation seemed to be similar to, but much lower than that of LEPAL-1. In the intact plant, both genes are expressed mainly in the root, the organ in which shikonin is exclusively produced and accumulated. Genomic Southern blot analyses showed that both genes are present in the genome as single copies.