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 QRFP receptor (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Didier Bagnol ◽  
Tom I. Bonner ◽  
Myrna Carlebur ◽  
Anthony P. Davenport ◽  
Stephen M. Foord ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cdna Library ◽  
Human Gene ◽  
Chromosome 1 ◽  
Chromosome 4 ◽  
Gene Encoding ◽  
Acid Protein ◽  
Sequence Similarities ◽  
Orphan Gpcr ◽  
Amino Acid Protein

The human gene encoding the QRFP receptor (nomenclature as agreed by the NC-IUPHAR Subcommittee on the QRFP receptor [16]; QRFPR, formerly known as the Peptide P518 receptor), previously designated as an orphan GPCR receptor was identified in 2001 by Lee et al. from a hypothalamus cDNA library [15]. However, the reported cDNA (AF411117) is a chimera with bases 1-127 derived from chromosome 1 and bases 155-1368 derived from chromosome 4. When corrected, QRFPR (also referred to as SP9155 or AQ27) encodes a 431 amino acid protein that shares sequence similarities in the transmembrane spanning regions with other peptide receptors. These include neuropeptide FF2 (38%), neuropeptide Y2 (37%) and galanin Gal1 (35%) receptors.

scholarly journals The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

1999 ◽  
Vol 65 (12) ◽  
pp. 5546-5553 ◽  
Author(s):  
Kazuhiro Iwashita ◽  
Tatsuya Nagahara ◽  
Hitoshi Kimura ◽  
Makoto Takano ◽  
Hitoshi Shimoi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Enzyme Assays ◽  
Partial Amino Acid Sequence ◽  
Reading Frame ◽  
Acid Protein ◽  
Aspergillus Kawachii ◽  
Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

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.

Molecular cloning, characterization, and expression analysis of a novel sema-2a homologue in Polyrhachis vicina (Hymenoptera: Formicidae)

2010 ◽  
Vol 142 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Jing Luo ◽  
Geng-Si Xi ◽  
Shu-Min Lü ◽  
Ke Li ◽  
Qing Li
Keyword(s):  
Untranslated Region ◽  
Axonal Guidance ◽  
Mrna Levels ◽  
Chain Reaction ◽  
Base Pairs ◽  
Reading Frame ◽  
Acid Protein ◽  
Polymerase Chain ◽  
Polyrhachis Vicina ◽  
Amino Acid Protein

AbstractThe semaphorin gene family plays important roles in axonal guidance in vertebrates and invertebrates. Semaphorin 2a, a member of the semaphorin family, belongs to class 2, which is found only in invertebrates. In our study, semaphorin 2a was cloned from the ant Polyrhachis vicina Roger. The full length of P. vicina semaphorin 2a (Pv-sema-2a) is 2763 base pairs (bp) and it contains a 5′-untranslated region (UTR) 92 bp long and a 3′-UTR 521 bp long. The open reading frame of Pv-sema-2a encodes a 716-amino-acid protein with a predicted molecular mass of 81.1 kilodaltons. Real-time quantitative reverse-transcription – polymerase chain reaction indicated that Pv-sema-2a mRNA is differentially expressed during P. vicina development, in the whole bodies as well as the heads of different castes. The high mRNA levels in embryos and pupae suggest that Pv-sema-2a plays an important role in ant development.

scholarly journals New Carbenicillin-Hydrolyzing β-Lactamase (CARB-7) from Vibrio cholerae Non-O1, Non-O139 Strains Encoded by the VCR Region of the V. cholerae Genome

2002 ◽  
Vol 46 (7) ◽  
pp. 2162-2168 ◽  
Author(s):  
Roberto Melano ◽  
Alejandro Petroni ◽  
Alicia Garutti ◽  
Héctor Alex Saka ◽  
Laura Mange ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Vibrio Cholerae ◽  
Gene Families ◽  
Chromosome 2 ◽  
Reading Frame ◽  
West Region ◽  
Acid Protein ◽  
Lactamase Gene ◽  
Flanking Regions ◽  
Amino Acid Protein

ABSTRACT In a previous study, an analysis of 77 ampicillin-nonsusceptible (resistant plus intermediate categories) strains of Vibrio cholerae non-O1, non-O139, isolated from aquatic environment and diarrheal stool, showed that all of them produced a β-lactamase with a pI of 5.4. Hybridization or amplification by PCR with a probe for bla TEM or primers for bla CARB gene families was negative. In this work, an environmental ampicillin-resistant strain from this sample, ME11762, isolated from a waterway in the west region of Argentina, was studied. The nucleotide sequence of the structural gene of the β-lactamase was determined by bidirectional sequencing of a Sau3AI fragment belonging to this isolate. The gene encodes a new 288-amino-acid protein, designated CARB-7, that shares 88.5% homology with the CARB-6 enzyme; an overall 83.2% homology with PSE-4, PSE-1, CARB-3, and the Proteus mirabilis N29 enzymes; and 79% homology with CARB-4 enzyme. The gene for this β-lactamase could not be transferred to Escherichia coli by conjugation. The nucleotide sequence of the flanking regions of the bla CARB-7 gene showed the occurrence of three 123-bp V. cholerae repeated sequences, all of which were found outside the predicted open reading frame. The upstream fragment of the bla CARB-7 gene shared 93% identity with a locus situated inside V. cholerae's chromosome 2. These results strongly suggest the chromosomal location of the bla CARB-7 gene, making this the first communication of a β-lactamase gene located on the VCR island of the V. cholerae genome.

Cloning and characterization of a 2,3-oxidosqualene cyclase from Eleutherococcus trifoliatus

Holzforschung ◽  
2013 ◽  
Vol 67 (4) ◽  
pp. 463-471 ◽  
Author(s):  
Li-Ting Ma ◽  
Sheng-Yang Wang ◽  
Yen-Hsueh Tseng ◽  
Yi-Ru Lee ◽  
Fang-Hua Chu
Keyword(s):  
Physiological Role ◽  
Active Site Residues ◽  
Reading Frame ◽  
Oxidosqualene Cyclases ◽  
Oxidosqualene Cyclase ◽  
Acid Protein ◽  
Leaf Tissues ◽  
New Perspective ◽  
Amino Acid Protein

Abstract The 2,3-oxidosqualene cyclases (OSCs) are a family of enzymes that have an important role in plant triterpene biosynthesis. In this study, an OSC gene designed EtLUS from Eleutherococcus trifoliatus has been cloned. EtLUS includes a 2292-bp open reading frame and encodes a 763-amino acid protein. EtLUS has an MLCYCR motif, which is conserved in lupeol synthases. Comparison of active-site residues and gene expression in yeast showed that EtLUS synthesizes lupeol. However, EtLUS has the highest sequence identity with β-amyrin synthases from Araliaceae rather than lupeol synthases, adding new perspective to the evolution of the OSCs of Araliaceae. Furthermore, EtLUS is upregulated in leaf tissues under methyl jasmonate treatment, which can be interpreted that lupeol and its derivatives play an ecological and physiological role in plant defense against pathogens and insect herbivores.

scholarly journals Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein

1993 ◽  
Vol 105 (3) ◽  
pp. 777-785 ◽  
Author(s):  
A.B. Vojtek ◽  
J.A. Cooper
Keyword(s):  
Adenylyl Cyclase ◽  
Leading Edge ◽  
Northern Analysis ◽  
Reading Frame ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Acid Protein ◽  
Carboxy Terminal ◽  
Amino Acid Protein

CAP, an adenylyl cyclase associated protein, is present in Saccharomyces cerevisiae and Schizosaccharomyces pombe. In both organisms, CAP is bifunctional: the N-terminal domain binds to adenylyl cyclase, thereby enabling adenylyl cyclase to respond appropriately to upstream regulatory signals, such as RAS in S. cerevisiae; the C-terminal domain is required for cellular morphogenesis. Here, we describe the isolation of a cDNA encoding a CAP homolog from a higher eukaryote. The mouse CAP cDNA contains an open reading frame capable of encoding a 474 amino acid protein. The protein encoded by the mouse CAP cDNA shows extensive homology to the yeast CAP proteins, particularly in the central poly-proline rich region and in the C-terminal domain. By northern analysis, the CAP message appears to be ubiquitous, but not uniform. By indirect immunofluorescence, ectopically expressed mouse CAP protein is found in the cytoplasm of fibroblasts and, in migrating cells, at the leading edge. Expression of the mouse CAP cDNA in S. cerevisiae complements defects associated with loss of the yeast CAP carboxy-terminal domain. Hence, the function of the CAP carboxy-terminal domain has been conserved from yeast to mouse.

scholarly journals Molecular Cloning and Characterization of a cDNA Encoding Sedoheptulose-1,7-bisphosphatase from Mulberry (Morus alba var. multicaulis)

2008 ◽  
Vol 57 (1-6) ◽  
pp. 152-157 ◽  
Author(s):  
X. Ji ◽  
Y. Gai ◽  
J. Ma ◽  
C. Zheng ◽  
Z. Mu
Keyword(s):  
Morus Alba ◽  
Evolutionary Analysis ◽  
Comparison Analysis ◽  
Reading Frame ◽  
Acid Protein ◽  
Fructose 1,6 Bisphosphatase ◽  
Rapid Amplification ◽  
Molecular Evolutionary Analysis ◽  
Amino Acid Protein

Abstract A full-length cDNA encoding sedoheptulose-1,7-bisphosphatase (SBPase; EC 3.1.3.37) was cloned from mulberry (Morus alba var. multicaulis) by rapid amplification of cDNA ends (RACE). The cDNA consisted of 1,527 nucleotides with an open reading frame (ORF) of 1,179 nucleotides encoding a 393 amino acid protein of approximately 42.6 kDa. Sequence comparison analysis showed that mulberry SBPase (MSBPase) had high homology to other plant counterparts. Phylogenetic and molecular evolutionary analysis revealed that MSBPase fell into plant SBPase group. Moreover, SBPase and fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) shared 28-32% identical residues, suggesting that the two enzymes originated from the same evolution branch. Molecular modeling indicated that each subunit of MSBPase was composed of α-helices and β-sheets joined by turns and loops, and folded into a structure of hexahedron shape which was very similar to FBPase.

scholarly journals Grtp1, A Novel Gene Regulated by Growth Hormone

Endocrinology ◽  
2001 ◽  
Vol 142 (10) ◽  
pp. 4568-4571 ◽  
Author(s):  
Chunxia Lu ◽  
John Kasik ◽  
Dietrich A. Stephan ◽  
Shaohua Yang ◽  
Mark A. Sperling ◽  
...  
Keyword(s):  
Northern Blot Analysis ◽  
Small Gtpases ◽  
Chromosome 8 ◽  
Taqman Assay ◽  
Activator Proteins ◽  
Novel Gene ◽  
Acid Protein ◽  
Vitro Model ◽  
Amino Acid Protein

Abstract An in vitro model of GH-responsive cells was subjected to microarray analysis to identify a novel gene regulated by GH. This 258 amino acid protein, we term GH Regulated TBC Protein-1 (GRTP1), contains the TBC signature motif of GTPase activator proteins of Rab-like small GTPases. Northern blot analysis revealed a 1.3 kb major mRNA species, most abundant in testes. TaqMan assay confirmed that in the mouse, Grtp1 is expressed at highest levels in testes, with lesser abundance in intestine, kidney, lung, and liver. In the testis, expression of Grtp1 significantly increases post-pubertally. Administration of GH to mice increased levels of GRTP1 mRNA in testes (140%), but decreased GRTP1 mRNA abundance in kidney (50%) and liver (25%). Grtp1 was localized to mouse proximal chromosome 8. Orthologs of this protein are present in human, mouse, rat, and drosophila suggesting that GRTP1 has an important biological role(s).

scholarly journals NOP3 is an essential yeast protein which is required for pre-rRNA processing.

1992 ◽  
Vol 119 (4) ◽  
pp. 737-747 ◽  
Author(s):  
I D Russell ◽  
D Tollervey
Keyword(s):  
Northern Analysis ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Rrna Processing ◽  
Reading Frame ◽  
Repeat Units ◽  
Nucleolar Proteins ◽  
Acid Protein ◽  
Sds Page ◽  
Amino Acid Protein

The four nucleolar proteins NOP1, SSB1, GAR1, and NSR1 of Saccharomyces cerevisiae share a repetitive domain composed of repeat units rich in glycine and arginine (GAR domain). We have cloned and sequenced a fifth member of this family, NOP3, and shown it to be essential for cell viability. The NOP3 open reading frame encodes a 415 amino acid protein with a predicted molecular mass of 45 kD, containing a GAR domain and an RNA recognition motif. NOP3-specific antibodies recognize a 60-kD protein by SDS-PAGE and decorate the nucleolus and the surrounding nucleoplasm. A conditional lethal mutation, GAL::nop3, was constructed; growth of the mutant strain in glucose medium represses NOP3 expression. In cells depleted of NOP3, production of cytoplasmic ribosomes is impaired. Northern analysis and pulse-chase labeling indicate that pre-rRNA processing is inhibited at the late steps, in which 27SB pre-rRNA is cleaved to 25S rRNA and 20S pre-rRNA to 18S rRNA.

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