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 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.

Molecular cloning and nucleotide sequence analysis of the putative cDNA for the precursor molecule of the chicken LH-β subunit

1989 ◽  
Vol 3 (2) ◽  
pp. 129-137 ◽  
Author(s):  
T. Noce ◽  
H. Ando ◽  
T. Ueda ◽  
K. Kubokawa ◽  
T. Higashinakagawa ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Cdna Clone ◽  
Rabbit Antiserum ◽  
Nucleotide Sequence Analysis ◽  
Cdna Expression Library ◽  
Β Subunit ◽  
Coding Region ◽  
Precursor Molecule ◽  
Cdna Insert ◽  
Hybridization Probe

ABSTRACT A cDNA expression library was constructed from poly(A)+ RNA of broiler chicken adenohypophyses using λ gt11 as a vector. After screening with a rabbit antiserum against chicken LH, a cDNA clone (L12) containing a 436 bp insert was obtained. Using a subclone of L12 in pUC19 (pL12) as the hybridization probe, another cDNA clone (LF127) with a 533 bp insert was isolated. The LF127 contained the full-length cDNA encoding the putative chicken LH-β subunit precursor molecule. Hybridization of the pL12 cDNA insert to adenohypophysial RNA showed that chicken and Japanese quail adenohypophyses contained RNA species of about 0·8 and 1·0 kb respectively. The amount of this RNA species was ten times higher in adult male quails kept under long days at room temperature than in those kept under short days at 7 °C. In-situ hybridization experiments showed the exclusive distribution of the signal in the LH cells of the adenohypophysis. The similarity of the nucleotide sequence of the apoprotein-coding region of LH-β cDNA of the chicken to that of mammals is lower than that among mammals. The deduced amino acid sequence of the chicken LH-β subunit supports the hypothesis that the number of proline residues increases in the LH-β subunit the closer phylogenetically the vertebrate is to mammals.

scholarly journals Acidic precursor revealed in human eosinophil granule major basic protein cDNA.

1988 ◽  
Vol 168 (4) ◽  
pp. 1493-1498 ◽  
Author(s):  
R L Barker ◽  
G J Gleich ◽  
L R Pease
Keyword(s):  
Endoplasmic Reticulum ◽  
Nucleotide Sequence ◽  
Cdna Library ◽  
Basic Protein ◽  
Cell Types ◽  
Major Basic Protein ◽  
Human Eosinophil ◽  
Damage Cell ◽  
Eosinophil Granule ◽  
Single Polypeptide

Eosinophil granule major basic protein (MBP), a potent toxin for helminths and various cell types, is a 13.8-kD single polypeptide rich in arginine with a calculated isoelectric point (pI) of 10.9. A cDNA for human MBP was isolated from a gamma GT10 HL-60 cDNA library. The nucleotide sequence of the MBP cDNA indicates that MBP is translated as a 25.2-kD preproprotein. The 9.9-kD pro-portion of proMBP is rich in glutamic and aspartic acids and has a calculated pI of 3.9, while proMBP itself has a calculated pI of 6.2. We suggest that MBP is translated as a nontoxic precursor that protects the eosinophil from damage while the protein is processed through the endoplasmic reticulum to its sequestered site in the granule core toxic MBP, and we present results from the literature suggesting that other cationic toxins, which damage cell membranes, may also be processed from nontoxic precursors containing distinct anionic and cationic regions.

scholarly journals Cloning and characterization of a second member of the mouse mdr gene family.

1988 ◽  
Vol 8 (7) ◽  
pp. 2770-2778 ◽  
Author(s):  
P Gros ◽  
M Raymond ◽  
J Bell ◽  
D Housman
Keyword(s):  
Multidrug Resistance ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Gene Family ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
Physiological Processes ◽  
Strong Homology ◽  
Energy Dependent

The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.

scholarly journals Biosynthesis of rat liver pI-6.1 esterase, a carboxylesterase of the cisternal space of the endoplasmic reticulum

1987 ◽  
Vol 248 (2) ◽  
pp. 545-550 ◽  
Author(s):  
M Robbi ◽  
H Beaufay
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Polyacrylamide Gel Electrophoresis ◽  
Rabbit Antiserum ◽  
Secretory Proteins ◽  
Structural Element ◽  
Oligosaccharide Moiety ◽  
Reticulocyte Lysate ◽  
Dog Pancreas ◽  
Processed Products

Biosynthesis of the rat liver microsomal esterase with pI 6.1 was investigated in cell-free systems and in cultured hepatocytes, by using a rabbit antiserum. Protein synthesis directed by total rat liver RNA in wheatgerm extract or reticulocyte lysate generated a single immunoprecipitable product, also found with the RNA extracted from bound, but not from free, polysomes. When dog pancreas microsomal fractions were included, reticulocyte lysates gave two processed products, a prominent one slightly larger, and another slightly smaller, than the precursor, both resistant to exogenous proteinases and, hence, segregated within vesicles. The processing was co-translational; it consisted of the removal of a peptide fragment and, for the large component, the addition of a single oligosaccharide chain. Indeed, this component bound to concanavalin A-Sepharose and gave the small one (approximately 2000 Mr loss) by cleavage with endo-beta-N-acetylglucosaminidase H (endo-H). A single labelled peptide was precipitated from hepatocytes incubated with [35S]methionine. Its apparent Mr was decreased by approximately 2000 after treatment with endo-H; it was then identical with that of an unglycosylated form produced in hepatocytes poisoned with tunicamycin. Even in that case, immunoreactive peptides were not detected in the culture medium. Whether synthesized in reticulocyte lysate or in hepatocytes, the glycosylated forms migrated in SDS/polyacrylamide-gel electrophoresis as the purified enzyme labelled with [3H]di-isopropyl fluorophosphate. Thus, although pI-6.1 esterase is not secreted, its biosynthesis is, as yet, indistinguishable from that of secretory proteins. Its oligosaccharide moiety is apparently not the structural element that retains it in the endoplasmic reticulum.

Complete characterization of the human IGF-I nucleotide sequence isolated from a newly constructed adult liver cDNA library

FEBS Letters ◽  
1986 ◽  
Vol 196 (1) ◽  
pp. 108-112 ◽  
Author(s):  
Y. Le Bouc ◽  
D. Dreyer ◽  
F. Jaeger ◽  
M. Binoux ◽  
P. Sondermeyer
Keyword(s):  
Nucleotide Sequence ◽  
Cdna Library ◽  
Complete Characterization ◽  
Adult Liver ◽  
Liver Cdna Library ◽  
Igf I

scholarly journals Expression of rat liver ketohexokinase in yeast results in fructose intolerance

1993 ◽  
Vol 291 (1) ◽  
pp. 179-186 ◽  
Author(s):  
I A Donaldson ◽  
T C Doyle ◽  
N Matas
Keyword(s):  
Mass Spectrometry ◽  
Amino Acid ◽  
Molecular Mass ◽  
Rat Liver ◽  
Amino Acid Sequences ◽  
Growth Media ◽  
Predicted Amino Acid Sequence ◽  
Sequence Information ◽  
Coding Region ◽  
Pcr Product

Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 2.7.1.3) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32,800 Da. The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24% of the total sequence, were determined. This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template. The 224 bp PCR product was used as a probe to screen a rat liver cDNA library. A cDNA sequence of 1342 bp was obtained from three positive clones. This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence. When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media. The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v). This response is associated with an accumulation of fructose 1-phosphate. The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da. This is in close agreement with the value for the isolated protein determined by mass spectrometry. The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokaryotic phosphotransferases that have furanose substrates.

Cloning and characterization of a second member of the mouse mdr gene family

1988 ◽  
Vol 8 (7) ◽  
pp. 2770-2778
Author(s):  
P Gros ◽  
M Raymond ◽  
J Bell ◽  
D Housman
Keyword(s):  
Multidrug Resistance ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Gene Family ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
Physiological Processes ◽  
Strong Homology ◽  
Energy Dependent

The mammalian mdr gene family comprises a small number of closely related genes. Previously, we have shown that one member, mdr1, has the capacity to convey multidrug resistance to drug-sensitive recipient cells in a gene transfer protocol. However, the functional characteristics of other members of this gene family have not been examined. In this report, we characterize a second member of the mdr gene family which we designated mdr2. We determined the nucleotide sequence corresponding to the complete coding region of this mdr2 transcript. The predicted amino acid sequence of this protein (1,276 amino acids) showed that it is a membrane glycoprotein highly homologous to mdr1 (85%), strongly suggesting that both genes originate from a common ancestor. Regions of divergence between mdr1 and mdr2 proteins are concentrated in two discrete segments of the predicted polypeptides, each approximately 100 residues in length. The mdr2 protein appears to be formed by the duplication of a structural unit which encodes three putative transmembrane loops and a predicted nucleotide-binding fold and is highly homologous to bacterial transport proteins such as hlyB. This strong homology suggests that mdr2 also participates in an energy-dependent membrane transport process. However, the direct relationship, if any, of this new member of the mdr family to multidrug resistance remains to be established. Knowledge of the complete nucleotide sequence and predicted amino acid sequence of the mdr2 gene product will enable the preparation of gene-specific probes and antibodies necessary to study the functional role of this gene in multidrug resistance and normal physiological processes.

Nucleotide sequence of the thioredoxin gene from Escherichia coli

1984 ◽  
Vol 4 (11) ◽  
pp. 917-923 ◽  
Author(s):  
Jan-Olov Höög ◽  
Hedvig von Bahr-Lindström ◽  
Staffan Josephson ◽  
Betty J. Wallace ◽  
Sidney R. Kushner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Restriction Enzyme ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
E Coli ◽  
Ribosome Binding ◽  
Sequence Method

The nucleotide sequence of the thioredoxin gene from Escherichia coli was determined. The structural gene was identified on a cloned 3-kb PvuII Iragment by hybridization with a synthetic oligodeoxyribonucleotide corresponding to a part of the amino acid sequence of thioredoxin. Restriction-enzyme fragments were used as templates in the dideoxy sequence method, directly and after subcloning into M13mp8. A segment of 450 nucleotides was determined using both strands7 alternatively, without extensive overlaps. The sequence contains the thioredoxin coding region, a potential ribosome-binding site, and a putative promotor region. The predicted amino acid sequence differs by two inversions from the previously given thioredoxin sequence. The revised sequence is presented and the results further show that thioredoxins from E. coli B and K12 are identical.

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