scholarly journals Cloning and expression of human liver dehydroepiandrosterone sulphotransferase

1993 ◽  
Vol 289 (1) ◽  
pp. 233-240 ◽  
Author(s):  
K A Comer ◽  
J L Falany ◽  
C N Falany
Keyword(s):  
Molecular Mass ◽  
Bile Acids ◽  
Blot Analysis ◽  
Human Liver ◽  
Molecular Size ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Acid Protein ◽  
Liver Cdna Library

Dehydroepiandrosterone sulphotransferase (DHEA-ST) catalyses the 3′-phosphoadenosine 5′-phosphosulphate-dependent sulphation of a wide variety of steroids in human liver and adrenal tissue and is responsible for most, if not all, of the sulphation of bile acids in human liver. This report describes the isolation, characterization and expression of a cDNA which encodes human liver DHEA-ST. The DHEA-ST cDNA, designated DHEA-ST8, was isolated from a Uni-Zap XR human liver cDNA library and is composed of 1060 bp and contains an open reading frame encoding a 285-amino-acid protein with a molecular mass of approx. 33765 Da. Translation of DHEA-ST8 in vitro generated a protein identical in molecular size with that of DHEA-ST. Expression of DHEA-ST8 in COS-7 cells produces an active DHEA-ST protein which is capable of sulphating DHEA, has the same molecular mass as human liver DHEA-ST and is recognized by rabbit anti-(human liver DHEA-ST) antibodies. Northern-blot analysis of human liver RNA detects the presence of three different size transcripts; however, Southern-blot analysis of human DNA suggests that only one gene may be present in the genome. These results describe the cloning of a human ST which has an important role in the sulphation of steroids and bile acids in human liver and adrenals.

scholarly journals Cloning and Expression of Two Novel Hemin Binding Protein Genes from Treponema denticola

2001 ◽  
Vol 69 (7) ◽  
pp. 4465-4472 ◽  
Author(s):  
Xiaoping Xu ◽  
Stanley C. Holt ◽  
David Kolodrubetz
Keyword(s):  
Blot Analysis ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Binding Activity ◽  
Cloning And Expression ◽  
Treponema Denticola ◽  
Reading Frame ◽  
E Coli ◽  
Significant Homology ◽  
Amino Acid Signal Peptide

ABSTRACT Treponema denticola does not appear to produce siderophores, so it must acquire iron by other pathways. Indeed,T. denticola has been shown to have an iron-regulated 44-kDa outer membrane protein (HbpA) with hemin binding ability. To characterize the HbpA protein, its gene was cloned from genomic DNA libraries of T. denticola. Sequence analysis of thehbpA open reading frame indicated that it encoded a 42.8-kDa protein with a 23-amino-acid signal peptide. HbpA has no significant homology to any proteins in the databases. Southern blot analysis demonstrated that hbpA is present in severalT. denticola ATCC strains and clinical isolates, but not inTreponema pectinovorum, Treponema socranskii, orEscherichia coli. HbpA, expressed as a recombinant protein in E. coli and purified by antibody affinity chromatography, has hemin binding activity as determined by lithium dodecyl sulfate-polyacrylamide gel electrophoresis with tetramethylbenzidine staining. Northern blot analysis showed that there were two hbpA-containing transcripts, of approximately 1.3 and 2.6 kb, and that the RNA levels were low-iron induced. Interestingly, the 2.6-kb mRNA also encoded a second protein with significant homology to hbpA. This downstream gene, calledhbpB, was cloned and sequenced and its product was expressed as a fusion protein in E. coli. ThehbpB gene product is 49% identical to HbpA and binds hemin. Thus, T. denticola has two novel hemin binding proteins which may be part of a previously unrecognized iron acquisition pathway.

scholarly journals Cloning and expression of a human choline/ethanolaminephosphotransferase: synthesis of phosphatidylcholine and phosphatidylethanolamine

1999 ◽  
Vol 339 (2) ◽  
pp. 291-298 ◽  
Author(s):  
Annette L. HENNEBERRY ◽  
Christopher R. McMASTER
Keyword(s):  
De Novo ◽  
Active Form ◽  
Fatty Acyl ◽  
Cloning And Expression ◽  
Amino Acid Residues ◽  
Reading Frame ◽  
Kennedy Pathway ◽  
Membrane Spanning

Cholinephosphotransferase catalyses the final step in the synthesis of phosphatidylcholine (PtdCho) via the Kennedy pathway by the transfer of phosphocholine from CDP-choline to diacylglycerol. Ethanolaminephosphotransferase catalyses an analogous reaction with CDP-ethanolamine as the phosphobase donor for the synthesis of phosphatidylethanolamine (PtdEtn). Together these two enzyme activities determine both the site of synthesis and the fatty acyl composition of PtdCho and PtdEtn synthesized de novo. A human choline/ethanolaminephosphotransferase cDNA (hCEPT1) was cloned, expressed and characterized. Northern blot analysis revealed one hCEPT1 2.3 kb transcript that was ubiquitous and not enriched, with respect to actin, in any particular cell type. The open reading frame predicts a protein (hCEPT1p) of 416 amino acid residues with a molecular mass of 46550 Da containing seven membrane-spanning domains. A predicted amphipathic helix resides within the active site of the enzyme with the final two aspartic residues of the CDP-alcohol phosphotransferase motif, DG(X)2AR(X)8G(X)3D(X)3D, positioned within this helix. hCEPT1p was successfully expressed in a full-length, active form in Saccharomyces cerevisiae cells devoid of endogenous cholinephosphotransferase or ethanolaminephosphotransferase activities (HJ091, cpt1::LEU2 ept1-). In vitro, hCEPT1p displayed broad substrate specificity, utilizing both CDP-choline and CDP-ethanolamine as phosphobase donors to a broad range of diacylglycerols, resulting in the synthesis of both PtdCho and PtdEtn. In vivo, S. cerevisiae cells (HJ091, cpt1::LEU2 ept1-) expressing hCEPT1 efficiently incorporated both radiolabelled choline and ethanolamine into phospholipids, demonstrating that hCEPT1p has the ability to synthesize both choline- and ethanolamine- containing phospholipids in vitro and in vivo.

Virus-encoded cyclin

1994 ◽  
Vol 14 (11) ◽  
pp. 7235-7244 ◽  
Author(s):  
J U Jung ◽  
M Stäger ◽  
R C Desrosiers
Keyword(s):  
Kinase Activity ◽  
Molecular Size ◽  
Protein Kinase Activity ◽  
Reading Frame ◽  
Kinase Activation ◽  
Type D ◽  
Cyclin Protein ◽  
High Level ◽  
Conserved Amino Acids

Herpesvirus saimiri contains an open reading frame called eclf2 with homology to the cellular type D cyclins. We now show that the eclf2 gene product is a novel virus-encoded cyclin (v-cyclin). The protein encoded by the v-cyclin gene of this oncogenic herpesvirus was found to have an apparent molecular size of 29 kDa in transformed cells. v-Cyclin protein was found to be associated with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins. cdk6/v-cyclin complexes strongly phosphorylated Rb fusion protein and histone H1 as substrates in vitro. Mutational analyses showed that highly conserved amino acids in the cyclin box of v-cyclin were important for association with cdk6 and for activation of cdk6 kinase activity. Thus, v-cyclin resembles cellular type D cyclins in primary sequence, in its association with cdk6, by its ability to activate protein kinase activity, and by the presence of functional cyclin box sequences. v-Cyclin exhibited a selective preference for association with cdk6 over other cyclin-dependent kinases and a high level of kinase activation. The properties of v-cyclin suggest a likely role in oncogenic transformation by this T-lymphotropic herpesvirus.

scholarly journals Virus-encoded cyclin.

1994 ◽  
Vol 14 (11) ◽  
pp. 7235-7244 ◽  
Author(s):  
J U Jung ◽  
M Stäger ◽  
R C Desrosiers
Keyword(s):  
Kinase Activity ◽  
Molecular Size ◽  
Protein Kinase Activity ◽  
Reading Frame ◽  
Kinase Activation ◽  
Type D ◽  
Cyclin Protein ◽  
High Level ◽  
Conserved Amino Acids

Herpesvirus saimiri contains an open reading frame called eclf2 with homology to the cellular type D cyclins. We now show that the eclf2 gene product is a novel virus-encoded cyclin (v-cyclin). The protein encoded by the v-cyclin gene of this oncogenic herpesvirus was found to have an apparent molecular size of 29 kDa in transformed cells. v-Cyclin protein was found to be associated with cdk6, a cellular cyclin-dependent kinase known to interact with cellular type D cyclins. cdk6/v-cyclin complexes strongly phosphorylated Rb fusion protein and histone H1 as substrates in vitro. Mutational analyses showed that highly conserved amino acids in the cyclin box of v-cyclin were important for association with cdk6 and for activation of cdk6 kinase activity. Thus, v-cyclin resembles cellular type D cyclins in primary sequence, in its association with cdk6, by its ability to activate protein kinase activity, and by the presence of functional cyclin box sequences. v-Cyclin exhibited a selective preference for association with cdk6 over other cyclin-dependent kinases and a high level of kinase activation. The properties of v-cyclin suggest a likely role in oncogenic transformation by this T-lymphotropic herpesvirus.

scholarly journals Molecular cloning, sequencing and expression studies of the human breast cancer cell glutaminase

2000 ◽  
Vol 345 (2) ◽  
pp. 365-375 ◽  
Author(s):  
Pedro M. GÓMEZ-FABRE ◽  
Juan C. ALEDO ◽  
Antonio DEL CASTILLO-OLIVARES ◽  
Francisco J. ALONSO ◽  
Ignacio NÚÑEZ DE CASTRO ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Breast Cancer ◽  
Human Liver ◽  
Molecular Size ◽  
Human Breast ◽  
Reading Frame ◽  
Rapid Amplification ◽  
Sequencing And Expression ◽  
Race Technique ◽  
Cell Growth And Proliferation

Phosphate-activated glutaminase (GA) is overexpressed in certain types of tumour but its exact role in tumour cell growth and proliferation is unknown. Here we describe the isolation of a full-length cDNA clone of human breast cancer ZR75 cells, by a combination of λgt10 cDNA library screening and the rapid amplification of cDNA ends (‘RACE’) technique. The cDNA of human GA is 2408 nt with a 1806-base open reading frame encoding a 602-residue protein with a predicted molecular mass of 66309 Da. The deduced amino acid sequence contains a putative mitochondrial import presequence of 14 residues at the N-terminal end. Heterologous expression and purification in Escherichia coli yielded a product of the expected molecular size that was recognized by using antibodies against the recombinant human GA. Sequence analyses showed that human GA was highly similar to the rat liver enzyme. Northern gel analysis revealed that the gene is present in human liver, brain and pancreas, in which a major transcript of 2.4 kb was demonstrated, but not in kidney, heart, skeletal muscle, lung or placenta. These results strongly suggest that the first human GA cloned, the GA from ZR-75 breast cancer cells, and presumably those from human liver and brain, are liver-type isoenzymes, in sharp contrast with the present view that considers the kidney type as the isoform expressed in all tissues with GA activity, with the exception of postnatal liver.

scholarly journals barS1, a Gene for Biosynthesis of a γ-Butyrolactone Autoregulator, a Microbial Signaling Molecule Eliciting Antibiotic Production in Streptomyces Species

2002 ◽  
Vol 184 (18) ◽  
pp. 5151-5157 ◽  
Author(s):  
Noriyasu Shikura ◽  
Junji Yamamura ◽  
Takuya Nihira
Keyword(s):  
Acyl Carrier Protein ◽  
Antibiotic Production ◽  
Receptor Gene ◽  
Streptomyces Species ◽  
Reading Frame ◽  
Streptomyces Virginiae ◽  
Acid Protein ◽  
Short Chain Alcohol ◽  
Natural Enantiomer

ABSTRACT From Streptomyces virginiae, in which production of streptogramin antibiotic virginiamycin M1 and S is tightly regulated by a low-molecular-weight Streptomyces hormone called virginiae butanolide (VB), which is a member of the γ-butyrolactone autoregulators, the hormone biosynthetic gene (barS1) was cloned and characterized by heterologous expression in Escherichia coli and by gene disruption in S. virginiae. The barS1 gene (a 774-bp open reading frame encoding a 257-amino-acid protein [M r, 27,095]) is situated in the 10-kb regulator island surrounding the VB-specific receptor gene, barA. The deduced BarS1 protein is weakly homologous to β-ketoacyl-acyl carrier protein/coenzyme A reductase and belongs to the superfamily of short-chain alcohol dehydrogenase. The function of the BarS1 protein in VB biosynthesis was confirmed by BarS1-dependent in vitro conversion of 6-dehydro-VB-A to VB-A, the last catalytic step in VB biosynthesis. Of the four possible enantiomeric products from racemic 6-dehydro-VB-A as a substrate, only the natural enantiomer of (2R,3R,6S)-VB-A was produced by the purified recombinant BarS1 (rBarS1), indicating that rBarS1 is the stereospecific reductase recognizing (3R)-isomer as a substrate and reducing it stereospecifically to the (6S) product. In the ΔbarS1 mutant created by homologous recombination, the production of VB as well as the production of virginiamycin was lost. The production of virginiamycin by the ΔbarS1 mutant was fully recovered by the external addition of VB to the culture, which indicates that the barS1 gene is essential in the biosynthesis of the autoregulator VBs in S. virginiae and that the failure of virginiamycin production was a result of the loss of VB production.

scholarly journals Molecular cloning and expression of a new putative inositol 1,4,5-trisphosphate 3-kinase isoenzyme

1991 ◽  
Vol 278 (3) ◽  
pp. 883-886 ◽  
Author(s):  
K Takazawa ◽  
J Perret ◽  
J E Dumont ◽  
C Erneux
Keyword(s):  
Amino Acid ◽  
Kinase Activity ◽  
Cloning And Expression ◽  
Fusion Product ◽  
Molecular Evidence ◽  
Reading Frame ◽  
Acid Protein ◽  
Sds Page ◽  
Inositol 1,4,5 Trisphosphate ◽  
Kinase A

A human hippocampus cDNA library in lambda ZAP II was screened by hybridization with a rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase cDNA. Two clones (hh6 and hh3) were isolated and sequenced. The insert of clone hh6 was shown to correspond to the 3′ end of the coding sequence of 50,000-Mr InsP3 3-kinase (referred to as 3-kinase-A). Sequencing of the clone hh3 insert yielded an open reading frame encoding a 472-amino acid protein with a calculated Mr of 53,451 (referred to as 3-kinase-B). The C-terminal part of 3-kinase-B (residues 187-462) was 68% identical with 3-kinase-A in amino acid sequence. The cDNA of clone hh3 was rescued as a Bluescript plasmid and expressed in Escherichia coli as a beta-galactosidase fusion product. It showed InsP3 3-kinase activity that was stimulated in the presence of Ca2+/calmodulin (more than 7-fold in a crude bacterial lysate from expressed plasmid). Regeneration of InsP3 3-kinase activity after SDS/PAGE identified a major polypeptide (Mr 60,000-65,000). The Km for InsP3 of expressed 3-kinase-B was 1.6 microM. These data provide molecular evidence for the existence of InsP3 3-kinase isoenzymes.

Cloning and expression of a novel UDP-GlcNAc:α-d-mannoside β1,2-N-acetylglucosaminyltransferase homologous to UDP-GlcNAc:α-3-d-mannoside β1,2-N-acetylglucosaminyltransferase I

2001 ◽  
Vol 361 (1) ◽  
pp. 153-162 ◽  
Author(s):  
Wenli ZHANG ◽  
Doron BETEL ◽  
Harry SCHACHTER
Keyword(s):  
Northern Blot Analysis ◽  
Human Gene ◽  
Cloning And Expression ◽  
Chromosome 1 ◽  
Tblastn Search ◽  
Reading Frame ◽  
Acid Protein ◽  
Human And Mouse ◽  
Amino Acid Protein

A TBLASTN search with human UDP-GlcNAc:α-3-d-mannoside β-1,2-N-acetylglucosaminyltransferase I (GnT I; EC 2.4.1.101) as a probe identified human and mouse Unigenes encoding a protein similar to human GnT I (34% identity over 340 amino acids). The recombinant protein converted Man(α1–6)[Man(α1–3)]Man(β1-)O-octyl to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl, the reaction catalysed by GnT I. The enzyme also added GlcNAc to Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1-)O-octyl (the substrate for β-1,2-N-acetylglucosaminyltransferase II), Man(α1-)O-benzyl [with Km values of ≈ 0.3 and > 30mM for UDP-GlcNAc and Man(α1-)O-benzyl respectively] and the glycopeptide CYA[Man(α1-)O-T]AV (Km ∼ 12mM). The product formed with Man(α1-)O-benzyl was identified as GlcNAc(β1–2)Man(α1-)O-benzyl by proton NMR spectroscopy. The enzyme was named UDP-GlcNAc:α-d-mannoside β-1,2-N-acetylglucosaminyltransferase I.2 (GnT I.2). The human gene mapped to chromosome 1. Northern-blot analysis showed a 3.3kb message with a wide tissue distribution. The cDNA has a 1980bp open reading frame encoding a 660 amino acid protein with a type-2 domain structure typical of glycosyltransferases. Man(β1-)O-octyl, Man(β1-)O-p-nitrophenyl and GlcNAc(β1–2)Man(α1–6)[GlcNAc(β1–2)Man(α1–3)]Man(β1–4)GlcNAc(β1–4)GlcNAc(β1-)O-Asn were not acceptors, indicating that GnT I.2 is specific for α-linked terminal Man and does not have N-acetylglucosaminyltransferase III, IV, V, VII or VIII activities. CYA[Man(α1-)O-T]AV was between three and seven times more effective as an acceptor than the other substrates, suggesting that GnT I.2 may be responsible for the synthesis of the GlcNAc(β1–2)Man(α1-)O-Ser/Thr moiety on α-dystroglycan and other O-mannosylated proteins.

scholarly journals Molecular characterization of a novel UT-A urea transporter isoform (UT-A5) in testis

2000 ◽  
Vol 279 (5) ◽  
pp. C1425-C1431 ◽  
Author(s):  
R. A. Fenton ◽  
A. Howorth ◽  
G. J. Cooper ◽  
R. Meccariello ◽  
I. D. Morris ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Molecular Size ◽  
Seminiferous Tubules ◽  
Mrna Levels ◽  
Putative Open Reading Frame ◽  
Reading Frame ◽  
Transporter Family ◽  
Acid Protein ◽  
Urea Uptake

Urea movement across plasma membranes is modulated by specialized transporter proteins that are products of two genes, termed UT-A and UT-B. These proteins play key roles in the urinary concentrating mechanism and fluid homeostasis. We have isolated and characterized a 1.4-kb cDNA from testes encoding a new isoform (UT-A5) belonging to the UT-A transporter family. For comparison, we also isolated a 2.0-kb cDNA from mouse kidney inner medulla encoding the mouse UT-A3 homologue. The UT-A5 cDNA has a putative open reading frame encoding a 323-amino acid protein, making UT-A5 the smallest UT-A family member in terms of molecular size. Its putative topology is of particular interest, because it calls into question earlier models of UT-A transporter structure. Expression of UT-A5 cRNA in Xenopus oocytes mediates phloretin-inhibitable urea uptake and does not translocate water. The distribution of UT-A5 mRNA is restricted to the peritubular myoid cells forming the outermost layer of the seminiferous tubules within the testes and is not detected in kidney. UT-A5 mRNA levels are coordinated with the stage of testes development and increase 15 days postpartum, commensurate with the start of seminiferous tubule fluid movement.

scholarly journals Expression of the bglH Gene ofLactobacillus plantarum Is Controlled by Carbon Catabolite Repression

1998 ◽  
Vol 180 (13) ◽  
pp. 3400-3404 ◽  
Author(s):  
Rosangela Marasco ◽  
Lidia Muscariello ◽  
Mario Varcamonti ◽  
Maurilio De Felice ◽  
Margherita Sacco
Keyword(s):  
Mutational Analysis ◽  
Glucose Repression ◽  
Transcriptional Unit ◽  
Calculated Molecular Mass ◽  
Reading Frame ◽  
Gene Coding ◽  
Acid Protein ◽  
Responsive Element

ABSTRACT A newly identified bglH gene coding for a phospho-β-glucosidase of Lactobacillus plantarum was isolated and expressed in Escherichia coli. The sequence analysis of the cloned DNA fragment showed an open reading frame encoding a 480-amino-acid protein with a calculated molecular mass of 53 kDa. The bglH gene was shown to be expressed on a monocistronic transcriptional unit. Its transcription was repressed 10-fold in L. plantarum cells grown on glucose compared to the β-glucoside salicin as a sole carbon source. A catabolite-responsive element (CRE) spanning from −3 to +11 with respect to the transcriptional start point was found, and its functionality was assessed by mutational analysis. In vitro and in vivo DNA binding experiments suggested the occurrence of a DNA-protein complex at the CRE site, which would mediate glucose repression ofbglH expression.

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