scholarly journals cDNA cloning, expression and chromosomal localization of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene

1996 ◽  
Vol 318 (2) ◽  
pp. 689-699 ◽  
Author(s):  
Leonard DODE ◽  
Frank WUYTACK ◽  
Patrick F. J. KOOLS ◽  
Fouzia BABA-AISSA ◽  
Luc RAEYMAEKERS ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Blot Hybridization ◽  
Cdna Probe ◽  
Human Platelets ◽  
Gene Transcript ◽  
Northern Blot Hybridization ◽  
Dna Encoding ◽  
Coding Region ◽  
Cos Cells

cDNA and genomic clones encoding human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) were isolated. The composite nucleotide sequence of the 4.6 kb cDNA, as well as the partial structure of 25 kb of genomic DNA encoding all but the 5´ region of the gene, was determined. The nucleotide sequence coding for the last six amino acids of the pump and the 3´-untranslated region were identified within the sequence of the last exon. Northern blot hybridization analysis using cDNA probes derived from this exon detected a 4.8 kb transcript in several human tissues. Using a cDNA probe derived from the 5´-coding region an unexpected mRNA distribution pattern, consisting of two mRNA species of 4.8 and 4.0 kb, was detected in thyroid gland and bone marrow only. This is the first indication of an alternative splicing mechanism operating on the SERCA3 gene transcript, which most likely generates SERCA3 isoforms with altered C-termini. Human SERCA3 expressed in platelets and in COS cells transfected with the corresponding cDNA was detected with the previously described antibody N89 (directed against the N-terminal region of rat SERCA3) and with a new SERCA3-specific antiserum C91, directed against the extreme C-terminus of the human isoform. A monoclonal antibody PL/IM430, previously assumed to recognize SERCA3 in human platelets, does not react with the 97 kDa human SERCA3 transiently expressed in COS cells. Therefore the 97 kDa isoform detected by PL/IM430 more likely represents a novel SERCA pump, as recently suggested [Kovács, Corvazier, Papp, Magnier, Bredoux, Enyedi, Sarkadi and Enouf (1994) J. Biol. Chem. 269, 6177–6184]. Finally, by fluorescence in situ hybridization and chromosome G-banding analyses, the SERCA3 gene was assigned to human chromosome 17p13.3.

Autocrine secretion of GM-CSF in acute myeloblastic leukemia

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1178-1181 ◽  
Author(s):  
DC Young ◽  
JD Griffin
Keyword(s):  
Blot Hybridization ◽  
Cdna Probe ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Acute Myeloblastic Leukemia ◽  
Leukemic Cells ◽  
Northern Blot Hybridization ◽  
In Vitro Proliferation ◽  
Gm Csf

Abstract Three cases of acute myeloblastic leukemia (AML) were identified in which clonogenic cells proliferated autonomously in vitro. Cells from two of these cases were found to secrete a colony-stimulating factor (CSF) that was immunologically and molecularly related to GM-CSF. Growth of AML-CFU could be blocked by the addition of a neutralizing antiserum to GM-CSF. Northern blot hybridization of leukemic cell mRNA with a cDNA probe for the GM-CSF gene revealed a 1-kb message identical in size to the normal GM-CSF message in stimulated T cells. No GM-CSF message was detected in the third case. These results indicate that constitutive expression of the GM-CSF gene, apparently by leukemic cells, can result in autonomous in vitro proliferation of AML-CFU in some cases of AML.

Post-transcriptional block in oxytocin gene expression within the seminiferous tubules of the bovine testis

1994 ◽  
Vol 140 (1) ◽  
pp. 63-72 ◽  
Author(s):  
H Ungefroren ◽  
M Davidoff ◽  
R Ivell
Keyword(s):  
Gene Expression ◽  
Leydig Cells ◽  
Blot Hybridization ◽  
Seminiferous Tubules ◽  
Transcriptional Level ◽  
Gene Transcript ◽  
Northern Blot Hybridization ◽  
Rnase Protection ◽  
Low Level

Abstract Northern blot hybridization showed that bovine and sheep testis, unlike testes from other mammals, contain moderate levels of an apparently normal oxytocin gene transcript. In situ hybridization localized this mRNA to within the seminiferous tubules, possibly in the Sertoli cells. Conflicting with this result, immunohistochemistry showed that both oxytocin and the syngeneic neurophysin I epitopes are both clearly restricted to the Leydig cells, being expressed here at a low level. Since illegitimate transcription from spurious start sites can lead to a lack of translation product, the integrity of the major ruminant testicular transcripts of the oxytocin gene was checked using differential hybridization, RNase protection and multiple polymerase chain reaction assays. All tests showed the transcripts to have a normal, translatable composition and to be transcribed from the conventional 5' initiation site. Therefore, the block in oxytocin gene expression within the tubules is probably due to a lesion at the post-transcriptional level. The low level peptide expression in the Leydig cells can probably be attributed to the presence of functional transcripts in these cells, which are below the level of significant detection for the in situ hybridization assay. Journal of Endocrinology (1994) 140, 63–72

scholarly journals Nucleotide sequence of cDNA coding for rat liver pI 6.1 esterase (ES-10), a carboxylesterase located in the lumen of the endoplasmic reticulum

1990 ◽  
Vol 269 (2) ◽  
pp. 451-458 ◽  
Author(s):  
M Robbi ◽  
H Beaufay ◽  
J N Octave
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Cdna Library ◽  
Rat Liver ◽  
Rabbit Antiserum ◽  
Protein Sequencing ◽  
Yeast Cells ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
Liver Cdna Library

A commercial rat liver cDNA library in lambda gt11 was screened with a rabbit antiserum to native pI 6.1 esterase (ES-10). The inserts of the immunoreactive clones were short (0.9-1.1 kbp). One of these was used as a probe to rescreen the library, yielding 30 clones, two of which contained relatively long (approx. 1.9 kbp) and widely overlapping cDNA inserts. They did not contain the first two nucleotide residues of the initiator codon, nor the 5′-end untranslated portion of the mRNA. These were derived from a home-made rat liver cDNA library in lambda gt11, screened with an oligonucleotide corresponding to the 5′-end of the already known cDNA sequence. The nucleotide sequence consists of 48 bp of 5′-end non-coding region, 1695 bp of coding region and 212 bp of 3′-end non-coding region including a 20 bp poly(A) tail. The signal peptide and the mature protein subunit are 18 and 547 residues long respectively. Tyr is confirmed as N-terminal residue. The predicted amino acid sequence is highly similar to those of rabbit liver esterase forms 1 (77% identity) and 2 (56% identity), determined by protein sequencing [Korza & Ozols (1988) J. Biol. Chem. 263, 3486-3495; Ozols (1989) J. Biol. Chem. 264, 12533-12545]. The three enzymes share the Ser and His residues presumed to be part of the active site, four Cys residues and a high proportion of charged side chains at their C-terminus. The C-terminal tetrapeptides of the three esterases (-HVEL, -HIEL and -HTEL for pI 6.1 and forms 1 and 2 esterases respectively) are reminiscent of, but not identical with, the localization signal identified in other proteins of the endoplasmic-reticulum lumen (-KDEL in animal cells [Munro & Pelham (1987) Cell 48, 899-907]; -HDEL in yeast [Pelham, Hardwick & Lewis (1988) EMBO J. 7, 1757-1762]). We still lack direct evidence to decide whether or not these C-terminal tetrapeptides commit esterases to reside in the endoplasmic reticulum. In that case the antepenultimate residue (D, V, I or T) would be only weakly stringent, and some sequences primed by H instead of K would be recognized in animal as well as in yeast cells.

scholarly journals Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1256-1262 ◽  
Author(s):  
FE Almus ◽  
LV Rao ◽  
UR Pendurthi ◽  
L Quattrochi ◽  
SI Rapaport
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Tissue Factor ◽  
Umbilical Vein ◽  
Blot Hybridization ◽  
Primary Cultures ◽  
Cdna Probe ◽  
Enzyme Linked Immunosorbent Assay ◽  
Northern Blot Hybridization ◽  
Heparin Binding

Abstract We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF- 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.

scholarly journals Autocrine secretion of GM-CSF in acute myeloblastic leukemia

Blood ◽  
1986 ◽  
Vol 68 (5) ◽  
pp. 1178-1181 ◽  
Author(s):  
DC Young ◽  
JD Griffin
Keyword(s):  
Blot Hybridization ◽  
Cdna Probe ◽  
Constitutive Expression ◽  
Leukemic Cell ◽  
Acute Myeloblastic Leukemia ◽  
Leukemic Cells ◽  
Northern Blot Hybridization ◽  
In Vitro Proliferation ◽  
Gm Csf

Three cases of acute myeloblastic leukemia (AML) were identified in which clonogenic cells proliferated autonomously in vitro. Cells from two of these cases were found to secrete a colony-stimulating factor (CSF) that was immunologically and molecularly related to GM-CSF. Growth of AML-CFU could be blocked by the addition of a neutralizing antiserum to GM-CSF. Northern blot hybridization of leukemic cell mRNA with a cDNA probe for the GM-CSF gene revealed a 1-kb message identical in size to the normal GM-CSF message in stimulated T cells. No GM-CSF message was detected in the third case. These results indicate that constitutive expression of the GM-CSF gene, apparently by leukemic cells, can result in autonomous in vitro proliferation of AML-CFU in some cases of AML.

scholarly journals Human platelets express the SERCA2-b isoform of Ca2+-transport ATPase

1992 ◽  
Vol 286 (1) ◽  
pp. 135-140 ◽  
Author(s):  
J Enouf ◽  
R Bredoux ◽  
B Papp ◽  
I Djaffar ◽  
A M Lompré ◽  
...  
Keyword(s):  
Cdna Library ◽  
Human Platelet ◽  
Cdna Probe ◽  
Human Cell Line ◽  
Human Platelets ◽  
Coding Region ◽  
Fast Skeletal Muscle ◽  
Biochemical Studies ◽  
Atpase System ◽  
Hel Cells

Previous biochemical studies suggested that the human platelet Ca2+ATPase system may be cell-specific. To test this hypothesis, we first undertook the molecular cloning of Ca2+ATPase from human erythroleukaemia (HEL) cells, because this human cell line exhibits megakaryocytic features and expresses a Ca2+ATPase that cross-reacts with platelet Ca(2+)-ATPase. For this cloning, an HEL-cell cDNA library was screened with a rat cardiac Ca2+ATPase cDNA probe. The insert of the longest clone isolated was 3.9 kb and its sequence displayed a 100% identity with that of the non-muscle human Ca2+ATPase 2-b isoform, termed SERCA2-b (sarco-endoplasmic-reticulum Ca2+ATPase). The 3.9 kb cDNA covered a subtotal coding region and part of the 3′ non-coding end of the SERCA2-b mRNA. It cross-hybridized with the 4 kb transcript species of cardiac SERCA2-a and with non-muscle SERCA2-b mRNAs, but not with fast-skeletal-muscle SERCA1 mRNA. We next confirmed that SERCA2-b was a component of the platelet Ca2+ATPase system because (1) the platelet clones isolated from a platelet cDNA library exhibited a 100% homology with HEL-cell cDNA; (2) SERCA2-b mRNA was amplified by PCR on total platelet RNA and (3) platelet Ca2+ATPase cross-reacted with a polyclonal SERCA2-b-specific antiserum. Platelets therefore contain a Ca2+ATPase definitely identified as the SERCA2-b isoform of Ca2+ATPase, thus eliminating the possibility that they only contain a single specific Ca2+ATPase.

scholarly journals Overexpression of eCLCA1 in Small Airways of Horses with Recurrent Airway Obstruction

2005 ◽  
Vol 53 (8) ◽  
pp. 1011-1021 ◽  
Author(s):  
Friederike Anton ◽  
Ina Leverkoehne ◽  
Lars Mundhenk ◽  
Wallace B. Thoreson ◽  
Achim D. Gruber
Keyword(s):  
Airway Obstruction ◽  
Blot Hybridization ◽  
Hek293 Cells ◽  
Sweat Glands ◽  
Northern Blot Hybridization ◽  
Small Airways ◽  
Recurrent Airway Obstruction ◽  
Nucleotide Polymorphisms ◽  
Coding Region

The human hCLCA1 and murine mCLCA3 (chloride channels, calcium-activated) have recently been identified as promising therapeutic targets in asthma. Recurrent airway obstruction in horses is an important animal model of human asthma. Here, we have cloned and characterized the first equine CLCA family member, eCLCA1. The 913 amino acids eCLCA1 polypeptide forms a 120-kDa transmembrane glycoprotein that is processed to an 80-kDa protein in vivo. Three single nucleotide polymorphisms were detected in the eCLCA1 coding region in 14 horses, resulting in two amino acid changes (485H/R and 490V/L). However, no functional differences were recorded between the channel properties of the two variants in transfected HEK293 cells. The eCLCA1 protein was detected immunohistochemically in mucin-producing cells in the respiratory and intestinal tracts, cutaneous sweat glands, and renal mucous glands. Strong overexpression of eCLCA1 was observed in the airways of horses with recurrent airway obstruction using Northern blot hybridization, Western blotting, immunohistochemistry, and real-time quantitative RT-PCR. The results suggest that spontaneous or experimental recurrent airway obstruction in horses may serve as a model to study the role of CLCA homologs in chronic airway disease with overproduction of mucins.

Transcriptional profiling of genes that are regulated by the endoplasmic reticulum-bound transcription factor AIbZIP/CREB3L4 in prostate cells

2007 ◽  
Vol 31 (2) ◽  
pp. 295-305 ◽  
Author(s):  
Sonia Ben Aicha ◽  
Julie Lessard ◽  
Mélissa Pelletier ◽  
Andréa Fournier ◽  
Ezequiel Calvo ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Transcription Factor ◽  
Blot Hybridization ◽  
Active Form ◽  
Physiological Role ◽  
Protein Processing ◽  
Protein Glycosylation ◽  
Bzip Transcription Factor ◽  
Calcium Stores ◽  
Northern Blot Hybridization

The androgen-regulated protein androgen-induced bZIP (AIbZIP) is a bZIP transcription factor that localizes to the membrane of the endoplasmic reticulum (ER). The physiological role of AIbZIP is unknown, but other ER-bound transcription factors such as ATF6 and SREBPs play a crucial role in the regulation of protein processing and lipid synthesis, respectively. In response to alterations in the intracellular milieu, ATF6 and SREBPs are processed to their transcriptionally active forms by regulated intramembrane proteolysis. In humans, AIbZIP mRNA is expressed in several organs including the pancreas, liver, and gonads, but it is especially abundant in prostate epithelial cells. We therefore used LNCaP human prostate cancer cells as a model to identify stimuli that lead to AIbZIP activation and define the transcriptional targets of AIbZIP. In LNCaP cells, AIbZIP was processed to its transcriptionally active form by drugs that deplete ER calcium stores (i.e., A23187 and caffeine), but it was unaffected by an inhibitor of protein glycosylation (tunicamycin). To identify AIbZIP-regulated genes, we generated LNCaP cell lines that conditionally express the processed form of AIbZIP and used Affymetrix microarrays to screen for AIbZIP-regulated transcripts. Selected genes ( n = 48) were validated by Northern blot hybridization. The results reveal that the downstream targets of AIbZIP include genes that are implicated in protein processing (e.g., BAG3, DNAJC12, KDELR3). Strikingly, a large number of AIbZIP-regulated transcripts encode proteins that are involved in transcriptional regulation, small molecule transport, signal transduction, and metabolism. These results suggest that AIbZIP plays a novel role in cell homeostasis.

scholarly journals Mechanism for diminished tissue factor expression by endothelial cells cultured with heparin binding growth factor-1 and heparin

Blood ◽  
1991 ◽  
Vol 77 (6) ◽  
pp. 1256-1262 ◽  
Author(s):  
FE Almus ◽  
LV Rao ◽  
UR Pendurthi ◽  
L Quattrochi ◽  
SI Rapaport
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Tissue Factor ◽  
Umbilical Vein ◽  
Blot Hybridization ◽  
Primary Cultures ◽  
Cdna Probe ◽  
Enzyme Linked Immunosorbent Assay ◽  
Northern Blot Hybridization ◽  
Heparin Binding

We have extended our earlier observation that growing primary cultures of human umbilical vein endothelial cells (HUVEC) with heparin binding growth factor 1 (HBGF-1) 20 micrograms/mL and heparin 12 U/mL inhibits expression of tissue factor (TF) activity on HUVC monolayers perturbed with thrombin. TF activity was measured as the ability of monolayers or cell lysates to support FVIIa-catalyzed activation peptide release from 3H-FX. TF antigen in HUVEC extracts was measured in an enzyme-linked immunosorbent assay (ELISA) that uses a double-antibody sandwich technique with rabbit and goat antibodies to human TF. TF-mRNA was measured by Northern blot hybridization with a 32P-TF cDNA probe. Cells growth with HBGF-1/heparin had both decreased surface and total TF activity as compared with HUVEC from the same endothelial cell pool grown without HBGF-1/heparin. Means +/- SD for TF antigen for four primary cultures were 4.4 +/- 0.9 ng/10(6) cells without HBGF-1/heparin and 0.6 +/- 0.3 ng/10(6) cells with HBGF-1/heparin. TF mRNA 4 hours after incubation with thrombin of HUVEC grown without HBGF-1/heparin was about sevenfold higher than TF mRNA of HUVEC grown with HBGF- 1/heparin. These data establish that growing primary cultures of HUVEC with HBGF-1/heparin impairs their ability to synthesize TF apoprotein after perturbation. This may be part of a generalized response of endothelial cells to HBGF-1/heparin facilitating migration during angiogenesis.

Sign in / Sign up

Export Citation Format

Share Document