cDNA cloning and characterization of a novel nucleolar protein.

In an initial study of anti-nuclear antibodies in the chronic inflammatory bladder disease interstitial cystitis, we reported that 7% of interstitial cystitis patients studied had autoantibodies to the nucleolus. We now report that, using an autoimmune serum from a patient with interstitial cystitis, we have identified and partially characterized a novel protein with an M(r) of approximately 55 kDa (hereafter referred to as No55) localized to the granular component of the nucleolus. No55 was initially characterized by diffuse nucleolar immunofluorescence staining in interphase cells and by Western blotting as a 55-kDa doublet on whole-cell extracts. During mitosis, No55 was associated with chromosomes and appeared in prenucleolar bodies during telophase, but it did not colocalize with p80-coilin in coiled bodies. Immunoelectron microscopy revealed that No55 was localized uniformly throughout the granular component of the nucleolus compared with a more peripheral localization of nucleolar granular component protein B23. On segregation of the nucleolus with actinomycin D, No55 remained with the granular component of the segregated nucleolus, whereas protein B23 was found predominantly in the nucleoplasm. Finally, a cDNA expression library was screened with the human autoantibody against No55, and a 2.4-kb insert was isolated, subcloned to homogeneity, and then sequenced. Analysis of this sequence showed an open reading frame of approximately 1.3 kb coding for 437 amino acids with a predicted molecular weight of 50 kDa. A search of the gene sequence database indicated homology with SC65, a rat synaptonemal complex protein. Therefore, on the basis of molecular weight, nucleolar sublocalization, response to actinomycin D, and cDNA sequence determination, No55 is a novel protein of the interphase nucleolus.     

Biochemical characterization, cDNA cloning and protein crystallization of aryl-alcohol oxidase from Pleurotus pulmonarius

Aryl-alcohol oxidase (AAO) involved in lignin degradation by Pleurotus pulmonarius has been purified and characterized. The enzyme was produced in glucose-peptone medium and isolated in a sole chromatographic step using Sephacryl S-200. The purified enzyme is an extracellular glycoprotein with 14% N-carbohydrate content and an estimated molecular mass of 70.5 kDa and pI of 3.95. The kinetic studies showed the highest enzyme affinity against p-anisyl alcohol, with constants similar to those of Pleurotus eryngii and Bjerkandera adusta AAO but different from the intracellular AAO described in Phanerochaete chrysosporium, which present the highest activity on m-anisyl alcohol. Simultaneously, the cDNA of P. pulmonarius AAO has been cloned and sequenced. The translation of this sequence consisted of 593 amino acids including a signal peptide of 27 amino acids. The comparison with other alcohol oxidases, 35% amino acid identity with glucose oxidase, showed highly conserved amino acid sequences in N-terminal and C-terminal regions, in spite of differences in substrate specificity. Crystallization of AAO, carried out for the first time using the P. pulmonarius enzyme, will permit to obtain a molecular model for this oxidase and establish some characteristic of its catalytic site and general structure.     

Purification, characterization, cDNA cloning and expression of a novel ketoreductase from Zygosaccharomyces rouxii.

A novel ketoreductase isolated from Zygosaccharomyces rouxii catalyzes the asymmetric reduction of selected ketone substrates of commercial importance. The 37.8-kDa ketoreductase was purified more than 300-fold to > 95% homogeneity from whole cells with a 30% activity yield. The ketoreductase functions as a monomer with an apparent Km for 3,4-methylenedioxyphenyl acetone of 2.9 mM and a Km for NADPH of 23.5 microM. The enzyme is able to effectively reduce alpha-ketolactones, alpha-ketolactams, and diketones. Inhibition is observed in the presence of diethyl pyrocarbonate, suggesting that a histidine is crucial for catalysis. The 1.0-kb ketoreductase gene was cloned and sequenced from a Z. rouxii cDNA library using a degenerate primer to the N-terminal sequence of the purified protein. Furthermore, it was expressed in both Escherichia coli and Pichia pastoris and shown to be active. Substrate specificity, lack of a catalytic metal, and extent of protein sequence identity to known reductases suggests that the enzyme falls into the carbonyl reductase enzyme class.     

cDNA cloning and characterization of a novel calmodulin-like protein from pearl oyster Pinctada fucata

Calcium metabolism in oysters is a very complicated and highly controlled physiological and biochemical process. However, the regulation of calcium metabolism in oyster is poorly understood. Our previous study showed that calmodulin (CaM) seemed to play a regulatory role in the process of oyster calcium metabolism. In this study, a full-length cDNA encoding a novel calmodulin-like protein (CaLP) with a long C-terminal sequence was identified from pearl oyster Pinctada fucata, expressed in Escherichia coli and characterized in vitro. The oyster CaLP mRNA was expressed in all tissues tested, with the highest levels in the mantle that is a key organ involved in calcium secretion. In situ hybridization analysis reveals that CaLP mRNA is expressed strongly in the outer and inner epithelial cells of the inner fold, the outer epithelial cells of the middle fold, and the dorsal region of the mantle. The oyster CaLP protein, with four putative Ca(2+)-binding domains, is highly heat-stable and has a potentially high affinity for calcium. CaLP also displays typical Ca(2+)-dependent electrophoretic shift, Ca(2+)-binding activity and significant Ca(2+)-induced conformational changes. Ca(2+)-dependent affinity chromatography analysis demonstrated that oyster CaLP was able to interact with some different target proteins from those of oyster CaM in the mantle and the gill. In summary, our results have demonstrated that the oyster CaLP is a novel member of the CaM superfamily, and suggest that the oyster CaLP protein might play a different role from CaM in the regulation of oyster calcium metabolism.     

Molecular cloning and characterization of a new cDNA encoding hyoscyamine 6beta-hydroxylase from roots of Anisodus acutangulus

A new full-length cDNA encoding hyoscyamine 6beta-hydroxylase (designated as aah6h, GenBank Accession No. EF187826), which catalyzes the last committed step in the scopolamine biosynthetic pathway, was isolated from young roots of Anisodus acutangulus by rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of aah6h was 1380 bp and contained a 1035 bp open reading frame (ORF) encoding a deduced protein of 344 amino acid residues. The deduced protein had an isoelectric point (pI) of 5.09 and a calculated molecular mass of about 38.7 kDa. Sequence analyses showed that AaH6H had high homology with other H6Hs isolated from some scopolamine-producing plants such as Hyoscyamus niger, Datura metel and Atropa belladonna etc. Bioinformatics analyses results indicated AaH6H belongs to 2-oxoglutarate-dependent dioxygenase superfamily. Phylogenetic tree analysis showed that AaH6H had closest relationship with H6H from A. tanguticus. Southern hybridization analysis of the genomic DNA revealed that aah6h belonged to a multi-copy gene family. Tissue expression pattern analysis firstly founded that aah6h expressed in all the tested tissues including roots, stems and leaves and indicated that aah6h was a constitutive-expression gene, which was the first reported tissue-independent h6h gene compared to other known h6h genes.     

cDNA cloning, identification and characterization of a novel cystatin from the tentacle of Cyanea capillata

Cystatin is of interest from biochemical and evolutionary prospective, and also has been applied in biotechnology. In this paper, a novel cystatin was found by EST sequence analysis of the cDNA library of Cyanea capillata tentacle. The sequence of a full-length cDNA clone contained an open reading frame encoding a putative 18-residue signal peptide and a mature protein of 113 amino acids, which showed only 26% identities to Family 2 cystatins and had its own characteristic enzyme-binding motifs, Ser(97)-Trp(98), which had not been found in any other known cystatins. Thus, the novel cystatin cloned from jellyfish was designated as cystatin J, which may belong to a new family of cystatin, called Family 4. The mature cystatin J was produced in Escherichia coli as a thioredoxin (Trx) fusion protein using the pET expression system and purified by affinity and cation exchange chromatography. The recombinant cystatin J of approximately M(r) = 12,800 displayed an obvious inhibition of papain (K(i) value below 0.5 nM), in competition with substrate. Thus, the recombinant cystatin J was a functional cystatin in spite of relatively lower sequence similarity with other cystatins. Activity of the novel cystatin was stable at pH 4-11 at 4 degrees C, but unstable at neutral pH at >50 degrees C.     

Purification, characterization, and cDNA cloning of profilin from Phaseolus vulgaris.

Profilin from common bean (Phaseolus vulgaris L.) was purified to homogeneity by poly-L-Pro affinity chromatography and gel filtration. The hypocotyl and symbiotic root nodule protein was detected as a single isoform with a 14.4-kD molecular mass and an isoelectric point of 5.3. Partial amino acid and DNA sequencing of a full-length cDNA clone confirmed its identity as profilin. An antibody generated against the purified protein binds to a protein with the same molecular mass in leaves and nodules. Immunolocalization of the protein showed a diffuse distribution in the cytoplasm of hypocotyls and nodules but enhanced staining at the vascular bundles. The strong identity of the sequence among the profilins of birch, maize, and bean suggests that it may play an important role in the signal transduction mechanism of plant cells and plant-bacterial symbioses.     

Molecular cloning and characterization of a cDNA encoding caltractin from Dunaliella salina.

We cloned a cDNA encoding caltractin, a 20 kDa calcium-binding protein, from Dunaliella salina (DSCALT). The Ca(2+)-bound mobility shift detected in Chlamydomonas caltractin was hardly detectable in DSCALT. Also, some differences were found in the electrophoretic mobility between the native DSCALT and the bacterial-expressed DSCALT. This difference may have resulted from the posttranslational modification. Immunoblot analysis revealed that this protein might be localized mainly in the basal body complex, the major microtubule organizing center (MTOC) in D.salina and the functional homologue of the centrosome of the animal cell.     

A procedure for isolating Pyrenochaeta terrestris from onion roots

Surface sterilised stelar tissues from onion roots exhibiting typical pink root symptoms were plated on water agar and incubated under cool white fluorescent light (CWFL) for 12 h day^-1 at 20°C. After 4–5 days, the plates were examined with the 10? objective of a compound microscope and tissues producing conidia of Fusarium spp. were discarded. Sub-cultures of isolates suspected to be Pyrenochaeta terrestris, grown from Fusarium-negative tissues, were transferred to chloroamphenicol-amended (500 ppm) corn-meal (CCMA) agar plates and incubated at 24°C under CWFL for a further 10–14 days. Cultures with setose pycnidia were identified as P. terrestris. Plates were again scanned for conidia of Fusarium spp., and cultures negative for Fusarium spp., but not producing pycnidia, were suspected to be P. terrestris. These were compared with known isolates on CCMA. On this medium growth of P. terrestris was slow, producing appressed, pinkish colonies which were circular with a smooth margin. All isolates of P. terrestris isolated by the procedure caused pink root of onion when tested, whereas none of the Fusarium spp. isolated produced the disease. However, a mixed inoculums of P. terrestris and Fusariurn solani produced typical pink root symptoms.     

Molecular cloning and characterization of type VII collagen cDNA.

Type VII collagen, located in human epidermal basement membrane, is the primary pathogenic target molecule in epidermolysis bullosa acquisita and epidermolysis bullosa dystrophica. Using a monoclonal antibody against the non-collagenous domain of type VII collagen, approximately 1 Kb cDNA was isolated from human keratinocyte library. The deduced primary structure of this clone thus reflects the non-collagenous domain of type VII collagen that may be involved in cell attachment. This region shows a weak homology (approximately 23%) to the cell attachment domain of fibronectin. Northern blot revealed approximately 9.5 Kb single band.     

Molecular cloning and characterization of rat estrogen receptor cDNA.

A cDNA clone of rat uterus estrogen receptor (ER) has been isolated and sequenced. This clone contains a complete open reading frame encoding 600 amino acid residues which is 5 and 11 amino acids larger than the corresponding molecules of human and chicken, respectively. The molecular weight of this protein is calculated to be 67,029. When this clone was ligated to the pSV2 vector and transfected into COS7 cells, a protein was produced that had the same affinity to estrogen as rat uterus ER. This sequence shows 88% homology with human ER; 528 amino acids are identical and 14 amino acids are conservative substitutions. The comparison of rat, human and chicken ER sequences indicate the presence of three highly conserved regions suggesting that these regions play important roles in ER function. The putative DNA-binding domain is completely identical in rat, human and chicken. The C-terminal half region which is thought to be the estrogen binding domain is also highly conserved and is rich in hydrophobic amino acid residues. Southern blot analysis of genomic DNA with ER cDNA as a probe has shown that related sequences are present in the genome.     

Postsynaptic short-chain neurotoxins from Pseudonaja textilis. cDNA cloning, expression and protein characterization.

Two lethal proteins, which specifically bind to the nAChR from Torpedo californica, were isolated from the venom of Pseudonaja textilis, the common brown snake from Australia. The isolated proteins have masses of 6236 and 6345 Da and are structurally related to short-chain neurotoxins from other elapids. Six cDNAs encoding isoforms of related neurotoxins were cloned using the RT-PCR of the venom gland mRNAs. The sequences of the corresponding proteins consist of 57-58 amino acid residues and display several unique features when compared with all known short-chain neurotoxins. Accordingly, they grouped separately in phylogenetic analysis. The six cDNAs were expressed in Escherichia coli and the recombinant proteins were characterized. They have similar masses and display similar toxicities and binding constants to the nAChR as the native toxins isolated from the venom. Thus, a new group of short-chain postsynaptic neurotoxins from the venom of an Australian elapid has been characterized.     

cDNA cloning and functional characterization of a novel splice variant of c-Cbl-associated protein from mouse skeletal muscle

c-Cbl-associated protein (CAP) is an SH3-containing adapter protein that binds to the proto-oncogene c-Cbl. Recent work suggests that signaling through these molecules is involved in the regulation of insulin-stimulated glucose uptake in 3T3-L1 adipocytes. Skeletal muscle is the major site of insulin-stimulated glucose disposal but there have been no reports of CAP function in this tissue. Using RT-PCR of mouse skeletal muscle RNA, we discovered a novel splice variant of CAP (CAPSM; GenBank Accession No. AF521593) that is different from the adipocyte form by inclusion of a novel 168 bp fragment. This fragment encodes a peptide sequence that shows very high similarity with exon 25 of the human homologue of CAP (SORBS1). To understand the function of CAPSM in glucose uptake regulation, L6 myotubes were transfected with either CAPSM or a truncated CAPSM devoid of all three SH3-binding domains (CAPDeltaSH3), which prevents CAP association with c-Cbl. Transfection with CAPDeltaSH3 decreased insulin-stimulated 2-deoxyglucose (2-DG) uptake and reduced c-Cbl phosphorylation. In contrast, transfection of L6 myotubes with CAPDeltaSH3 had no effect on dinitrophenol (DNP)- or hypoxia-stimulated glucose uptake, stimuli that work through insulin-independent mechanisms for the regulation of glucose uptake. These data demonstrate the existence of a novel CAP isoform expressed in skeletal muscle, and suggest the involvement of the CAP/Cbl pathway in the regulation of insulin-stimulated glucose uptake in L6 myotubes.     

Purification, characterization, and cDNA cloning of a novel metallothionein-like, cadmium-binding protein from Caenorhabditis elegans

Caenorhabditis elegans adapted for survival in high concentrations of Cd(II) express a heavy metal binding protein designated C. elegans metallothionein-like protein or MT-Ce. This protein was purified to homogeneity and characterized. MT-Ce binds 6 mol of Cd(II)/mol protein. The sequence of 39 amino-terminal residues in MT-Ce was determined. A radiolabeled 41-mer oligonucleotide, designed from the partial MT-Ce sequence, was used in conjunction with sucrose gradient centrifugation to obtain size-fractionated poly(A+) RNA enriched in MT-Ce sequences. Subsequently, cloned cDNAs, corresponding to MT-Ce mRNA sequences, were isolated from a lambda ZapII cDNA library prepared from the enriched template mRNA. cDNA and protein sequence analysis revealed that MT-Ce comprises 62 amino acid residues and has a predicted Mr of 6462. Seventeen of the 18 Cys residues in the nematode cadmium-binding protein are included in Cys-X-Cys and X-Cys-Cys-X motifs that are characteristic of mammalian metallothioneins (MTs). However, the resemblance of MT-Ce to mammalian MTs is superficial. The amino acid sequence of MT-Ce is unique, and neither its putative alpha and beta domains nor its Cys residues can be readily aligned with the corresponding regions of other eukaryotic MTs. This suggests that MT-Ce is an example of convergent evolution. The MT-Ce mRNA level in nematodes that were selected and grown with Cd(II) concentrations that are lethal for wild-type worms, was 55-fold higher than the level of MT-Ce mRNA in wild-type C. elegans. Comparison of the sequences of MT-Ce cDNAs revealed the occurrence of two types of MT-Ce mRNA. Each contains an identical coding region, but the cDNAs diverge markedly in their 5'-untranslated regions. This suggests the possibilities of regulation by alternative splicing and/or the presence of multiple MT-Ce genes encoding a single protein, but controlled by different regulatory elements.     

cDNA cloning and characterization of vinculin mRNA

Vinculin and meta-vinculin are related proteins that localize to adherens junctions. To facilitate studies on the biology of these proteins cDNA clones were isolated from a chick embryonic library made in lambda gt 11. Three clones, accounting for 1/2 of the vinculin message, were identified by immunological analyses of fusion proteins and by hybrid-arrested translation. Sequence analyses of the 3' ends of these clones reveal allelic variants. Northern blot analyses suggests the presence of multiple vinculin messages, the most prominent one at 6.2 kb, almost twice the size needed to encode vinculin.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.