scholarly journals Evidence that the 14 kDa soluble β-galactoside-binding lectin in man is encoded by a single gene

1989 ◽  
Vol 259 (1) ◽  
pp. 291-294 ◽  
Author(s):  
W M Abbott ◽  
T Feizi
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Cell ◽  
Cdna Clone ◽  
Human Placenta ◽  
Southern Blotting ◽  
Single Gene ◽  
Cdna Probe ◽  
Human Cell Lines ◽  
Binding Lectin

A full-length cDNA clone for the 14 kDa soluble beta-galactoside-binding lectin of man has been isolated from a cDNA library from HepG2 hepatoma cells. The derived amino acid sequence is identical with that of the 14 kDa lectin from human placenta. The results of Northern and Southern blotting of several different human cell lines using a cDNA probe for the 14 kDa lectin suggest the presence of a single gene for this protein. Thus, although there are multiple proteins in the range 14-200 kDa which are antigenically related to this lectin, we would conclude from the present study that there is only one gene for the 14 kDa lectin.

scholarly journals Human myeloperoxidase gene: molecular cloning and expression in leukemic cells

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1411-1414
Author(s):  
KS Chang ◽  
JM Trujillo ◽  
RG Cook ◽  
SA Stass
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Clone ◽  
Northern Blot ◽  
Single Gene ◽  
Myelogenous Leukemia ◽  
Cloning And Expression ◽  
Leukemic Cells ◽  
Expression Library

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity- purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

scholarly journals The sequence of the mouse 14 kDa β-galactoside-binding lectin and evidence for its synthesis on free cytoplasmic ribosomes

1989 ◽  
Vol 261 (3) ◽  
pp. 847-852 ◽  
Author(s):  
T J G Wilson ◽  
M N Firth ◽  
J T Powell ◽  
F L Harrison
Keyword(s):  
Amino Acid ◽  
Basement Membrane ◽  
Amino Acid Sequence ◽  
Genomic Dna ◽  
Cdna Probe ◽  
Cdna Clones ◽  
Partial Amino Acid Sequence ◽  
3T3 Fibroblasts ◽  
Cytoplasmic Ribosomes ◽  
Binding Lectin

The partial amino acid sequence of the mouse 14 kDa beta-galactoside-binding lectin has been deduced from cDNA clones corresponding to 86% of the coding sequence and extending to the polyadenylation signal. The deduced amino acid sequence for the murine lectin shows 94% identity with the rat, 89% with human, 86% with bovine and 46% with the chicken 14 kDa lectins. A cDNA probe has been used to analyse genomic DNA and identify a single mRNA of approx. 570 bp in 3T3 fibroblasts, murine erythroleukaemia cells and the murine basement-membrane-secreting Engelbreth-Holm-Swarm tumour. Analysis of free and bound polyribosomes has shown that the lectin message is translated on free cytoplasmic ribosomes.

scholarly journals Human myeloperoxidase gene: molecular cloning and expression in leukemic cells

Blood ◽  
1986 ◽  
Vol 68 (6) ◽  
pp. 1411-1414 ◽  
Author(s):  
KS Chang ◽  
JM Trujillo ◽  
RG Cook ◽  
SA Stass
Keyword(s):  
Gene Expression ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Clone ◽  
Northern Blot ◽  
Single Gene ◽  
Myelogenous Leukemia ◽  
Cloning And Expression ◽  
Leukemic Cells ◽  
Expression Library

Abstract We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity- purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.

Identification and cDNA cloning of a Xenopus nucleolar phosphoprotein, xNopp180, that is the homolog of the rat nucleolar protein Nopp140

1995 ◽  
Vol 108 (10) ◽  
pp. 3339-3347 ◽  
Author(s):  
C. Cairns ◽  
B. McStay
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Cloning ◽  
Cdna Clone ◽  
Xenopus Oocyte ◽  
Ribosome Biogenesis ◽  
Nucleolar Protein ◽  
Nuclear Localisation Signal ◽  
Cdc2 Kinase

The monoclonal antibody G1C7, recognises both Xenopus nucleolin and a protein of 180 kDa present in Xenopus oocyte nucleoli. This antibody was used to obtain a cDNA clone encoding the 180 kDa protein now called xNopp180 (Xenopus nucleolar phosphoprotein of 180 kDa). Analysis of the deduced amino acid sequence from this cDNA shows that xNopp180 is almost entirely composed of alternating acidic and basic domains. We show that xNopp180 is heavily phosphorylated and that it contains multiple consensus sites for phosphorylation by casein kinase II and cdc2 kinase. In addition we show that xNopp180 is the 180 kDa antigen recognised by the monoclonal antibody No-114, thus allowing reinterpretation of previous work with this antibody. xNopp180 appears to be the Xenopus homolog of the rat nucleolar protein Nopp140. Nopp140 is a nuclear localisation signal binding protein that shuttles on curvilinear tracks between the nucleolus and the cytoplasm. Possible roles for xNopp180/Nopp140 in ribosome biogenesis are discussed.

REGULATION OF PLASMINOGEN ACTIVATOR INHIBITOR-1 mRNA IN HUMAN ENDOTHELIAL CELLS

1987 ◽  
Author(s):  
E A van den Berg ◽  
E Sprengers ◽  
M Jaye ◽  
W Burgess ◽  
V W M van Hinsbergh
Keyword(s):  
Endothelial Cells ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Plasminogen Activator ◽  
Cdna Clone ◽  
Plasminogen Activator Inhibitor ◽  
Mrna Levels ◽  
Human Endothelial Cells ◽  
Activator Inhibitor ◽  
Pai 1

Cultured human endothelial cells (HEC) increase their production of plasminogen activator inhibitor (PAI-1) upon stimulation with endotoxin and IL-1, agents that are known to cause an increase in PAI-1 levels in vivo. In order to study the regulation of PAI-1 synthesis at the mRNA level, we isolated a cDNA clone for the human PAI-1 gene from an endothelial expression cDNA library in λ gt 11 by screening with a PAI-1 specific antibody. Three positive cross-hybridizing clones were isolated. The longest insert (1500 bp) was partially sequenced (1000 bp). The sequence was identical to the PAI-1 sequence recently reported by others. The identity of the cDNA clone was further confirmed by comparison with part of the amino acid sequence of PAI-1. For that purpose t-PA-PAI-1 complex was purified from HEC conditioned medium by immunoadsorption to anti-t-PA IgG, and a suitable peptide was sequenced after comparison of the HPLC elution profiles of CNBr digests of t-PA and t-PA-PAI-1 complex. The amino acid sequence (M)FRQFQADFT completely matches the sequence predicted from the cDNA sequence.By hybridization of the cDNA probe to Northern blots of total cellular RNA from human umbilical vein and artery EC (HUVEC, HUAEC), two transcripts of 2.3 and 3 kb were found. Primary HUAEC, incubated for 18 hours in growth medium, produced considerable although variable levels of PAI-1 activity and contained PAI-1 mRNA levels comparable to those found in subcultured HUAEC. When subcultured HUEC were incubated for 6 h with endotoxin, IL-1 or TNF, a 2-fold increase in PAI-1 mRNA was found with each of these mediators. Stimulation of the cells in the presence of cycloheximide resulted in a further increase of the 3 kb PAI-1 transcript. The 3’ end of this transcript contains a 75 bp AT-rich sequence. Similar 3’ AT-rich sequences have been found in mRNA’s for a number of inflammatory mediators and cellular oncogenes, and in some cases it has been shown that removal of the sequence increased mRNA stability. The influence of cyclohex-imid on the larger PAI-1 transcript might be explained by inhibition of synthesis of a specific nuclease that controls the level of mRNA’s harbouring such an AT rich sequence.

scholarly journals Partial amino acid sequence and mRNA analysis of cytosolic pyridoxine-beta-d-glucoside hydrolase from porcine intestinal mucosa: proposed derivation from the lactase-phlorizin hydrolase gene

2004 ◽  
Vol 380 (1) ◽  
pp. 211-218 ◽  
Author(s):  
Chi-Wah TSEUNG ◽  
Laura G. McMAHON ◽  
Jorge VÁZQUEZ ◽  
Jan POHL ◽  
Jesse F. GREGORY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Northern Blot ◽  
Sequence Data ◽  
Cdna Probe ◽  
Protein Mass ◽  
Sds Page ◽  
Mrna Analysis ◽  
Hydrolysis Of

We have previously identified and purified a novel β-glucosidase, designated PNGH (pyridoxine-5´-β-d-glucoside hydrolase), from the cytosolic fraction of pig intestinal mucosal. PNGH catalyses the hydrolysis of PNG (pyridoxine-5´-β-d-glucoside), a plant derivative of vitamin B6 that exhibits partial nutritional bioavailability in humans and animals. Preliminary amino acid sequence analysis indicated regions of close similarity of PNGH to the precursor form of LPH (lactase–phlorizin hydrolase), the β-glucosidase localized to the brush-border membrane. We report in the present study amino acid sequence data for PNGH and results of Northern blot analyses, upon which we propose a common genomic origin of PNGH and LPH. Internal Edman sequencing of the PNGH band isolated by SDS/PAGE yielded data for 16 peptides, averaging 10.8 amino acids in length. These peptides from PNGH (approx. 140 kDa) were highly similar to sequences existing over most of the length of the >200 kDa precursor of rabbit LPH; however, we found no PNGH sequences that corresponded to approx. 350 amino acids between positions 463 and 812 of the LPH precursor, a region encoded by exon 7 of the LPH precursor gene (amino acids 568–784), and no sequences that corresponded to regions near the N-terminus. MS analysis of tryptic peptides yielded 25 peptides, averaging 15 amino acids, with masses that matched segments of the rabbit LPH precursor. Northern blot analysis of pig and human small intestinal polyadenylated mRNA using a non-specific LPH cDNA probe showed an expected approx. 6 kb transcript of the LPH precursor, but also an approx. 4 kb transcript that was consistent with the size predicted from the PNGH protein mass. Using a probe specific to the region encoded by exon 7, hybridization occurred only with the 6 kb transcript. Based on these observations, we propose that both PNGH and LPH enzymes have the same genomic origin, but differ in transcriptional and, possibly, post-translational processing.

Nucleotide and predicted amino acid sequence of a cDNA clone encoding part of human transketolase

1992 ◽  
Vol 183 (3) ◽  
pp. 1159-1166 ◽  
Author(s):  
Mahin Abedinia ◽  
Roy Layfield ◽  
Sheelagh M. Jones ◽  
Peter F. Nixon ◽  
John S. Mattick
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Clone ◽  
Predicted Amino Acid Sequence
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