scholarly journals Cloning and expression of a cDNA encoding a mouse brain orphanin FQ/nociceptin precursor

1996 ◽  
Vol 315 (1) ◽  
pp. 11-13 ◽  
Author(s):  
Ying-Xian PAN ◽  
Jin XU ◽  
Gavril W. PASTERNAK
Keyword(s):  
Amino Acid ◽  
Mouse Brain ◽  
Single Gene ◽  
Amino Acid Level ◽  
Cloning And Expression ◽  
Northern Analysis ◽  
Orphanin Fq ◽  
Amino Acid Peptide ◽  
Reverse Transcription Pcr ◽  
Mouse Sequence

By using a reverse transcription–PCR approach we have cloned a peptide precursor from mouse brain which contains the sequence of orphanin FQ/nociceptin. The mouse sequence of orphanin FQ/nociceptin is identical at the amino acid level with that isolated from rat and porcine brain. Northern analysis of the mRNA encoding the precursor reveals a single band of approx. 1 kb, with the highest levels in the brain and much lower levels in kidney and spleen. Southern analysis is consistent with a single gene. The precursor peptide from mouse contains two other putative peptides. Upstream from the orphanin FQ/nociceptin is a 41-amino-acid peptide which is almost identical, except for a six-amino-acid insertion, with the corresponding 35-amino-acid peptide predicted from the rat sequence. Interestingly, the mouse contains a triple AEPGAD repeat within this peptide that is not seen in the rat sequence. Downstream from the orphanin FQ/nociceptin sequence is another 17-amino-acid peptide which is identical with that found in the rat.

scholarly journals Neuromedin U receptors (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Khaled Al-hosaini ◽  
Stephen R. Bloom ◽  
Joseph Hedrick ◽  
Andrew Howard ◽  
Preeti Jethwa ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Bone Marrow ◽  
Amino Acid ◽  
Species Differences ◽  
Fetal Liver ◽  
Single Gene ◽  
Acid Product ◽  
Amino Acid Peptide ◽  
Upper Gastrointestinal ◽  
Sole Product

Neuromedin U receptors (provisional nomenclature as recommended by NC-IUPHAR [29]) are activated by the endogenous 25 amino acid peptide neuromedin U (neuromedin U-25, NmU-25), a peptide originally isolated from pig spinal cord [90]. In humans, NmU-25 appears to be the sole product of a precursor gene (NMU, P48645) showing a broad tissue distribution, but which is expressed at highest levels in the upper gastrointestinal tract, CNS, bone marrow and fetal liver. Much shorter versions of NmU are found in some species, but not in human, and are derived at least in some instances from the proteolytic cleavage of the longer NmU. Despite species differences in NmU structure, the C-terminal region (particularly the C-terminal pentapeptide) is highly conserved and contains biological activity. Neuromedin S (neuromedin S-33) has also been identified as an endogenous agonist [95]. NmS-33 is, as its name suggests, a 33 amino-acid product of a precursor protein derived from a single gene and contains an amidated C-terminal heptapeptide identical to NmU. NmS-33 appears to activate NMU receptors with equivalent potency to NmU-25.

scholarly journals Cloning and Characterization of the Vitamin D Receptor from Xenopus laevis*

Endocrinology ◽  
1997 ◽  
Vol 138 (6) ◽  
pp. 2347-2353 ◽  
Author(s):  
Yan Chun Li ◽  
Clemens Bergwitz ◽  
Harald Jüppner ◽  
Marie B. Demay
Keyword(s):  
Vitamin D ◽  
Amino Acid ◽  
Xenopus Laevis ◽  
Vitamin D Receptor ◽  
Messenger Rna ◽  
Mammalian Cells ◽  
Amino Acid Level ◽  
Ion Homeostasis ◽  
Northern Analysis ◽  
Lower Vertebrate

Abstract The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor α, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (preneurulation), increasing to maximum at stages 57–61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.

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.

Cloning, sequencing and expression of stem cell factor (c-kit ligand) cDNA of brushtail possum (Trichosurus vulpecula)

1996 ◽  
Vol 8 (4) ◽  
pp. 789 ◽  
Author(s):  
PJ Greenwood ◽  
C Seamer ◽  
DJ Tisdall
Keyword(s):  
Stem Cell ◽  
Amino Acid ◽  
Stem Cell Factor ◽  
Biological Activities ◽  
Amino Acid Level ◽  
Amino Acid Sequences ◽  
Northern Analysis ◽  
Nucleotide Sequencing ◽  
Brushtail Possum ◽  
Factor C

By means of reverse transcription polymerase chain reaction (RT-PCR), three stem cell factor (SCF) cDNAs (822-738 bp in size) were amplified from brushtail possum ovarian poly (A)+ RNA. The largest and smallest of these cDNAs were cloned and sequenced. Characterization of these cDNAs has revealed that possum SCF has approximately 75% and 66% homology to SCF of eutherian mammals at the nucleotide level and the predicted amino acid level respectively. Nucleotide sequencing shows that the 738-bp cDNA represents an mRNA splice variant, equivalent to that found in eutherian mammals, in which an exon (84 bp) encoding a potential proteolytic cleavage site is removed. Comparison of the predicted possum SCF amino acid sequence with the predicted SCF amino acid sequences from eutherian mammals reveals conservation of all cysteine residues and 3 of 4 potential N-linked glycosylation sites. In addition, the hydropathicity profile of the possum SCF protein is similar to that of eutherian SCF suggesting that protein conformation is conserved. Northern analysis was used to characterize possum SCF gene expression in adult ovary and testis. A major transcript of 9 kb was observed in both ovarian and testicular tissue. The conservation of the SCF gene and its predicted protein, suggests that SCF in the possum has similar biological activities to SCF in eutherian mammals.

scholarly journals Molecular characterisation and hormone-dependent expression of the porcine whey acidic protein gene

1998 ◽  
Vol 20 (1) ◽  
pp. 27-35 ◽  
Author(s):  
KJ Simpson ◽  
P Bird ◽  
D Shaw ◽  
K Nicholas
Keyword(s):  
Gene Expression ◽  
Mammary Gland ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Level ◽  
Whey Acidic Protein ◽  
Acidic Protein ◽  
Northern Analysis ◽  
Major Protein ◽  
Sds Page

A 17.5 kDa protein was isolated from porcine whey by reverse phase HPLC and identified as a putative whey acidic protein (WAP) homologue by sequencing 35 and 40 amino acid residues of the amino- and carboxy-terminus respectively. Degenerate oligonucleotides to both of these amino acid sequences were designed and used in reverse transcriptase PCR with RNA from lactating porcine mammary gland as a template. A 162 bp PCR fragment was detected and sequenced. Compilation of the deduced and determined amino acid sequence revealed a protein of 111 amino acids, which had approximately 75, 50, 40 and 35% similarity at amino acid level to camel, rabbit, rat and mouse WAP respectively. It also included the four-disulphide core characteristic of all WAP proteins and most Kunitz-type protease inhibitors. This provides the first unequivocal evidence for WAP secretion in the pig. SDS PAGE analysis of the whey fraction showed that WAP is secreted as a major protein in sow's milk from farrowing to weaning. The molecular mass of WAP in SDS PAGE was significantly greater than the 11.7 kDa determined from amino acid sequence, indicating that porcine WAP is possibly glycosylated. Northern analysis detected a single mRNA transcript of approximately 600 bp in porcine RNA from the mammary gland of lactating sows. To examine the hormone-regulated expression of the WAP gene the mammary glands of sows at day 90 of pregnancy were biopsied and explants cultured for 3 days in the presence of various combinations of porcine insulin (I), cortisol (F) and porcine prolactin (P). Northern analysis of RNA extracted from the tissue indicated that WAP gene expression was barely detectable in the mammary gland prior to culture and there was no increment in explants cultured in the presence of I and F. However, a significant increase in the accumulation of WAP mRNA was observed in explants cultured in I, F and P. A similar result was observed for beta-casein and alpha-lactalbumin gene expression.

scholarly journals Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase.

1990 ◽  
Vol 10 (6) ◽  
pp. 2678-2686 ◽  
Author(s):  
G P Livi ◽  
P Kmetz ◽  
M M McHale ◽  
L B Cieslinski ◽  
G M Sathe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Gene Product ◽  
Cell Function ◽  
Single Gene ◽  
Antidepressant Drug ◽  
Cloning And Expression ◽  
Cdna Clones ◽  
Additional Function ◽  
Recombinant Gene

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

Cloning and expression of cDNA for a human low-Km, rolipram-sensitive cyclic AMP phosphodiesterase

1990 ◽  
Vol 10 (6) ◽  
pp. 2678-2686
Author(s):  
G P Livi ◽  
P Kmetz ◽  
M M McHale ◽  
L B Cieslinski ◽  
G M Sathe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Cyclic Amp ◽  
Gene Product ◽  
Cell Function ◽  
Single Gene ◽  
Antidepressant Drug ◽  
Cloning And Expression ◽  
Cdna Clones ◽  
Additional Function ◽  
Recombinant Gene

We have isolated cDNA clones representing cyclic AMP (cAMP)-specific phosphodiesterases (PDEases) from a human monocyte cDNA library. One cDNA clone (hPDE-1) defines a large open reading frame of ca. 2.1 kilobases, predicting a 686-amino-acid, ca. 77-kilodalton protein which contains significant homology to both rat brain and Drosophila cAMP PDEases, especially within an internal conserved domain of ca. 270 residues. Amino acid sequence divergence exists at the NH2 terminus and also within a 40- to 100-residue domain near the COOH-terminal end. hPDE-1 hybridizes to a major 4.8-kilobase mRNA transcript from both human monocytes and placenta. The coding region of hPDE-1 was engineered for expression in COS-1 cells, resulting in the overproduction of cAMP PDEase activity. The hPDE-1 recombinant gene product was identified as a low-Km cAMP phosphodiesterase on the basis of several biochemical properties including selective inhibition by the antidepressant drug rolipram. Known inhibitors of other PDEases (cGMP-specific PDEase, cGMP-inhibited PDEase) had little or no effect on the hPDE-1 recombinant gene product. Human genomic Southern blot analysis suggests that this enzyme is likely to be encoded by a single gene. The presence of the enzyme in monocytes may be important for cell function in inflammation. Rolipram sensitivity, coupled with homology to the Drosophila cAMP PDEase, which is required for learning and memory in flies, suggests an additional function for this enzyme in neurobiochemistry.

scholarly journals Cloning and Expression of a Xylitol-4-Dehydrogenase Gene from Pantoea ananatis

2006 ◽  
Vol 72 (1) ◽  
pp. 368-377 ◽  
Author(s):  
J. S. Aarnikunnas ◽  
A. Pihlajaniemi ◽  
A. Palva ◽  
M. Leisola ◽  
A. Nyyssölä
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Northern Analysis ◽  
Nucleotide Sequence Analysis ◽  
Pantoea Ananatis ◽  
Reading Frame ◽  
Recombinant Cells ◽  
Dehydrogenase Gene

ABSTRACT The Pantoea ananatis ATCC 43072 mutant strain is capable of growing with xylitol as the sole carbon source. The xylitol-4-dehydrogenase (XDH) catalyzing the oxidation of xylitol to l-xylulose was isolated from the cell extract of this strain. The N-terminal amino acid sequence of the purified protein was determined, and an oligonucleotide deduced from this peptide sequence was used to isolate the xylitol-4-dehydrogenase gene (xdh) from a P. ananatis gene library. Nucleotide sequence analysis revealed an open reading frame of 795 bp, encoding the xylitol-4-dehydrogenase, followed by a 5′ region of another open reading frame encoding an unknown protein. Results from a Northern analysis of total RNA isolated from P. ananatis ATCC 43072 suggested that xdh is transcribed as part of a polycistronic mRNA. Reverse transcription-PCR analysis of the transcript confirmed the operon structure and suggested that xdh was the first gene of the operon. Homology searches revealed that the predicted amino acid sequence of the P. ananatis XDH shared significant identity (38 to 51%) with members of the short-chain dehydrogenase/reductase family. The P. ananatis xdh gene was successfully overexpressed in Escherichia coli, XDH was purified to homogeneity, and some of its enzymatic properties were determined. The enzyme had a preference for NAD+ as the cosubstrate, and in contrast to previous reports, the enzyme also showed a side activity for the d-form of xylulose. Xylitol was converted to l-xylulose with a high yield (>80%) by the resting recombinant cells, and the l-xylulose was secreted into the medium. No evidence of d-xylulose being synthesized by the recombinant cells was found.

scholarly journals Cloning and Expression of ntnD, Encoding a Novel NAD(P)+-Independent 4-Nitrobenzyl Alcohol Dehydrogenase from Pseudomonas sp. Strain TW3

2000 ◽  
Vol 182 (11) ◽  
pp. 3136-3141 ◽  
Author(s):  
Keith D. James ◽  
Michelle A. Hughes ◽  
Peter A. Williams
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Benzyl Alcohol ◽  
Cloning And Expression ◽  
Significant Similarity ◽  
Reverse Transcription Pcr ◽  
Nitrobenzyl Alcohol ◽  
Dehydrogenase Gene

ABSTRACT Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)+ independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)+-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.

Deorphanization of ORL-1/LC132 by reverse pharmacology in two landmark studies

2018 ◽  
Author(s):  
Mark F. Bird ◽  
David G. Lambert
Keyword(s):  
Nervous System ◽  
Neuropathic Pain ◽  
Amino Acid ◽  
G Protein Coupled Receptors ◽  
Peptide Ligands ◽  
Orphanin Fq ◽  
Amino Acid Peptide ◽  
Endogenous Peptide ◽  
G Protein Coupled ◽  
Intracellular Inhibition

Deorphanization of ORL-1/LC132 in 1995 by reverse pharmacology in two simultaneously published landmark studies added a new member to the opioid family of G-protein coupled receptors. Meunier and Reinscheid used cells expressing recombinant ORL-1 (human) or LC132 (rat) and the presumed intracellular inhibition of cyclic AMP formation to ‘fish’ for endogenous peptide ligands in rat whole-brain and pig hypothalamic extracts. Both studies reported the isolation of a 17-amino-acid peptide, which was named nociceptin and orphanin FQ by the two authors, respectively. The behaviour of the isolated peptide was a complete surprise, as a general hyperalgesia was observed when the peptide was administered at supraspinal sites. We now know that this peptide has, in fact, anti-opioid action, particularly in the medulla. The endogenous peptide exerts a multitude of effects both in the nervous system and, unlike classical opioids, has efficacy in neuropathic pain.

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