Cloning and sequence analysis of the cDNA for the rat atrial natriuretic factor precursor

Atrial extracts contain factors which induce potent natriuresis changes in renal haemodynamics, and relax pre-contracted vascular smooth muscle. Low-molecular-weight peptides which mimic these actions have now been purified by several groups, including ours (see accompanying paper), and higher-molecular-weight proteins with similar but less potent biological activities have also been identified and are presumed to be precursors. If released into the circulation, these peptides, collectively called atrial natriuretic factor (ANF), may play a significant part in blood-pressure homeostasis, regulation of extracellular fluid volume and as antagonists to the hypertensive effects of the renin-angiotensin system and other hormonal and neurotransmitter systems. We describe here the isolation and characterization of rat atrial cDNA clones which encode ANF. Nucleotide sequence analysis shows that auriculin corresponds to the 25 amino acids located close to the C-terminus of a 152-amino acid ANF precursor. Analysis of the in vitro translation products of precursor ANF mRNA suggests that multiple forms of the precursor may exist.     

Nucleotide sequence of the gene encoding human atrial natriuretic factor precursor

The mammalian atrium is an endocrine organ that may be involved in the control of blood pressure and extracellular fluid volume. A series of peptides, which seem to be associated with atrium-specific secretory granules, have potent natriuretic, diuretic and smooth muscle relaxant activities. Sequence determination of several of these peptides, which range from 21 to 126 amino acids long, shows that they form a family, derived from a common precursor. Rat and human complementary DNAs that encode the precursor to the various peptides, collectively called atrial natriuretic factors (ANFs), have been cloned. Nucleotide sequencing showed that the ANFs are located at the C-terminus of a polypeptide of relative molecular mass 13,000. We describe here the isolation and characterization of the corresponding human gene. Two introns interrupt the gene; one is located in the region coding for the N-terminus of the precursor and the other separates the codon for the C-terminal tyrosine from the rest of the peptide.     

Cloning and sequence analysis of cDNA encoding a precursor for human atrial natriuretic polypeptide

Recent identification of natriuretic-diuretic activity in peptides isolated from human and rat atrial tissue implicates them in the control of extracellular fluid volume and electrolytic homeostasis. The presence of multiple forms of the peptides ranging from 3,000 to 13,000 molecular weight (MW) suggests they may all derive from the same precursor. The established amino acid sequence of alpha-human atrial natriuretic polypeptide (alpha- hANP ), a 28-residue peptide with potent natriuretic activity, provided the means to elucidate the structure of the precursor for alpha- hANP and the gene encoding it. Here we report the cloning and sequence analysis of the cDNA of human atrial mRNA encoding a precursor of alpha- hANP . The cDNA encodes gamma-human atrial natriuretic polypeptide (gamma- hANP ) of 13,000 MW, whose C-terminal 28 amino acid residues may be processed as alpha- hANP .     

Primary structure of alpha-subunit precursor of Torpedo californica acetylcholine receptor deduced from cDNA sequence

DNA sequences complementary to the Torpedo californica electroplax mRNA coding for the alpha-subunit precursor of the acetylcholine receptor were cloned. The nucleotide sequence of the cloned cDNA indicates that the precursor consists of 461 amino acids including a prepeptide of 24 amino acids. Possible sites for acetylcholine binding and antigenic determinants on the alpha-subunit molecule are discussed.     

mRNA sequence for human cardiodilatin-atrial natriuretic factor precursor and regulation of precursor mRNA in rat atria

The mammalian cardiac atrium has recently been shown to contain numerous peptides that exert marked effects on kidney function and vascular resistance. With one exception, the peptides have potent natriuretic and diuretic activities, and sequence similarities suggest that they may be derived from a common atrial natriuretic factor (ANF) precursor. The exception, cardiodilatin (CDD), differs from the ANF peptides in possessing potent vasorelaxant activity, but not natriuretic and diuretic activities. Here we report the cloning and sequence analysis of the cDNA for the human precursor protein (preproCDD-ANF) containing both the CDD and ANF sequences. The CDD sequence represents the N-terminal sequence preceded directly by a signal peptide, while the ANF sequence is present at the C-terminal end of the protein. Using hybridization analysis, we further show that the amount of rat preproCDD-ANF mRNA, which is synthesized selectively in the atria but not the ventricles, markedly decreases on water deprivation, suggesting that the water-electrolyte balance may be an important factor in the regulation of the expression of the preproCDD-ANF gene.     

Primary structure of Torpedo californica acetylcholinesterase deduced from its cDNA sequence

Acetylcholinesterase, an essential enzyme of the nervous system, rapidly terminates the action of acetylcholine released into the synapse. Acetylcholinesterase is also found (in lower abundance) in extrajunctional areas of muscle and nerve and on erythrocyte membranes. Hydrodynamic analyses of the native enzyme and characterization of its dissociated subunits have revealed multiple enzyme forms which can be divided into two classes: dimensionally asymmetric forms which are usually found within the synapse and contain a collagen-like structural subunit disulphide-linked to the catalytic subunits; and globular forms which appear to be widely distributed on the outer surface of cell membranes. Both forms have been characterized in the ray Torpedo californica and, although their catalytic behaviours seem to be identical, they differ slightly in amino-acid composition, peptide maps and reactivity with certain monoclonal antibodies. Here, we report the complete amino-acid sequence of an acetylcholinesterase inferred from the sequence of a complementary DNA clone. The 575-residue protein shows significant homology with the C-terminal portion of thyroglobulin.     

Deduced amino acid sequence from the bovine oxytocin-neurophysin I precursor cDNA

The nonapeptide hormone oxytocin-like arginine-vasopressin (AVP) is synthesized as part of a larger precursor polypeptide. The precursor also includes the neurophysin molecule with which the hormone is associated in the neurosecretory granules of the hypothalamo-pituitary tract. A protein of molecular weight (Mr) approximately 20,000 has been isolated from supraoptic nuclei of rat hypothalami which, after tryptic cleavage, released a neurophysin-like molecule of Mr approximately 10,000 and an oligopeptide related to oxytocin. This result was complemented by in vitro translation of bovine hypothalamic mRNA. Among the primary translation products a single polypeptide of Mr approximately 16,500 was shown to contain antigenic determinants recognized by specific antisera against bovine neurophysin I and oxytocin. Here we report the amino acid sequence of the bovine oxytocin-neurophysin I (OT-NpI) precursor which was derived from sequence analysis of the cloned cDNA. As is the case for the bovine arginine-vasopressin-neurophysin II (AVP-NpII) precursor, the signal sequence of the OT-NpI precursor is immediately followed by the nonapeptide hormone which is connected to neurophysin I by a Gly-Lys-Arg sequence. A striking feature of the nucleic acid sequence is the 197-nucleotide long perfect homology with the AVP-NpII precursor mRNA sequence encoding the conserved middle part of neurophysins I and II.     

Nucleotide sequence of epidermal growth factor cDNA predicts a 128,000-molecular weight protein precursor

Epidermal growth factor (EGF) has a profound effect on the differentiation of specific cells in vivo, and has been shown to be a potent mitogenic factor for a variety of cultured cells, of both ectodermal and mesodermal origin (see ref. 1 for review). This 53-amino acid polypeptide of known sequence contains six cysteine residues, which are thought to form three intrachain disulphide bonds. Urogastrone, a polypeptide bearing anti-gastric secretory activity isolated from human urine, which is presumably synthesized in submandibular and Brunner's glands, shares extensive sequence homology (70%) with EGF and may represent the human EGF equivalent. Here we present the sequence of a mouse EGF cDNA clone, which suggests that EGF is synthesized as a large protein precursor of 1,168 amino acids. Our data indicate that the discrepancy between EGF levels in male and female mouse submaxillary glands (MSGs) is due to different EGF mRNA levels in these tissues, and suggest that precursor EGF processing may differ from that described previously for other polypeptide hormones.     

Nucleotide sequence of cloned cDNA for bovine corticotropin-beta-lipotropin precursor.

The nucleotide sequence of a 1,091-base pair cloned cDNA insert encoding bovine corticotropin-beta-lipotropin precursor mRNA is reported. The corresponding amino acid sequence indicates that the precursor protein consists of repetitive units and includes a third melanotropin sequence in its cryptic portion. Pairs of lysine and arginine residues separate the component peptides of the precursor.     

Characterization of cDNA and genomic clones encoding the precursor to rat hypothalamic growth hormone-releasing factor

Growth hormone-releasing factor (GHRF) is a hypothalamic peptide which positively regulates the synthesis and secretion of growth hormone in the anterior pituitary. The amino-acid sequence of a 43-residue GHRF peptide isolated from rat hypothalamus was recently determined. Immunocytochemical techniques have been used to localize GHRF-containing cell bodies and nerve fibres largely to the medial-basal region of the rat hypothalamus. The rat has also been used extensively as an animal model to study the effects of GHRF on growth hormone synthesis and secretion and on somatic growth. To pursue questions concerning the biosynthesis of GHRF, the expression of the ghrf gene, and its regulation in the hypothalamus by neural and hormonal influences, we have now isolated and characterized both complementary DNA and genomic clones encoding rat hypothalamic GHRF. The rat ghrf gene spans nearly 10 kilobases (kb) of rat genomic DNA, contains 5 exons and encodes a 104-amino-acid precursor to the rat GHRF peptide. Comparison with previously characterized human ghrf cDNA and genomic clones has allowed patterns of conservation of amino-acid and nucleotide sequences between the human and rat GHRFs to be determined.     

Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA

The sodium/potassium-dependent ATPase [(Na+ + K+)ATPase], which establishes and maintains the Na+ and K+ gradients across the plasma membrane of animal cells, consists of two subunits, alpha and beta. Complementary DNA clones encoding the catalytic (alpha) subunit of sheep kidney and Torpedo californica electroplax enzymes have previously been isolated and characterized. However, there is little information concerning the primary structure of the beta-subunit, a glycoprotein of unknown function and relative molecular mass (Mr) approximately 55,000 (ref. 3). Here we describe the isolation and characterization of a cDNA clone containing the entire coding region of the beta-subunit of the sheep kidney (Na+ + K+)ATPase. We also discuss structural aspects of the protein and present evidence for a possible evolutionary relationship with the KdpC subunit of the Escherichia coli K+-ATPase.     

Nucleotide sequence of cloned cDNA encoding bovine arginine vasopressin-neurophysin II precursor

The sequence of a cDNA encoding the nonapeptide arginine vasopressin (AVP) and its carrier protein, neurophysin II (NpII) from bovine hypothalamus, proves that the 166-amino acid precursor molecule contains a signal peptide of 19 amino acids followed directly by AVP connected to NpII by a Gly-Lys-Arg sequence. The carboxy-terminal region of the precursor contains a naturally occurring glycopolypeptide of 39 amino acids which is separated from NpII by a single arginine residue.     

Sequence of cDNA encoding human insulin-like growth factor I precursor

Somatomedins (SM) or insulin-like growth factors (IGF) constitute a heterogeneous group of peptides with important growth-promoting effects in vitro as well as in vivo. Amino acid sequences have been determined for only two of them, IGF-I and IGF-II, which are highly homologous. IGF-I, which is identical with SM-C, is composed of 70 amino acid residues and IGF-II contains 73 amino acids and may be identical with SM-A. Other peptides with different charge properties but with similar SM-like or insulin-like behaviour in biological and receptor assays, have been described but have not yet been fully characterized. The liver is known to be a major site of production of these peptides, but many other tissues--especially in the fetus--may synthesize them as well. We report here the nucleotide sequence of a human liver cDNA encoding the complete amino acid sequence of IGF-I. The IGF-I coding region is flanked by sequences encoding an amino-terminal peptide of at least 25 amino acid residues and a carboxyl-terminal peptide of 35 amino acids. This provides evidence that IGF-I is synthesized as a precursor protein and that formation of IGF-I from this precursor requires proteolytic processing at both ends.     

Isolation of a cDNA clone encoding rat insulin-like growth factor-II precursor

Insulin-like growth factor-I (IGF-I) and IGF-II are mitogenic polypeptides of relative molecular mass (Mr) approximately 7,500 isolated from human plasma each containing four peptide domains in a single chain and identical at more than 60% of their amino acid loci. The B- and A-domains of the IGFs are approximately 40% identical to the B- and A-chains of human insulin. IGF-I and IGF-II have similar in vitro biological activities and receptor reactivity, but are immunologically distinct. IGF-I appears to mediate the effects of growth hormone on cartilage to promote skeletal growth whereas IGF-II may have a special role in fetal development and in the central nervous system. To investigate the in vivo role of IGF-II, we have studied IGF-II biosynthesis in the BRL-3A rat liver cell line. BRL-3A cells synthesize and secrete a 7,484 Mr protein 93% identical to human IGF-II and representing rat IGF-II (rIGF-II). Rat IGF-II is synthesized as a approximately 22,000 Mr prepro-rIGF-II (ref. 12) from 12 S poly(A)+mRNA. In addition, approximately 20,000 Mr pro-rIGF-II has been identified in lysates of biosynthetically labelled intact BRL-3A cells. We report here the isolation of an almost complete cDNA clone for rIGF-II. Our results indicate that pro-rIGF-II is synthesized as a 156 amino acid peptide precursor (17,619 Mr) containing mature rIGF-II 1-67 at its amino-terminus and an 89-residue carboxy-terminal peptide extension.     

Complete primary structure of a heterodimeric T-cell receptor deduced from cDNA sequences

Two related, but distinct, cDNA clones have been isolated and sequenced from a functional murine cytotoxic T-lymphocyte clone. The genes corresponding to these cDNA are expressed and rearranged specifically in T cells and both have similarities to immunoglobulin variable and constant region genes. It is concluded that these genes code for the two subunits of the heterodimeric antigen receptor on the surface of the T cell; its complete deduced primary structure is presented.     

Inhibition of aldosterone production in the adrenal glomerulosa by atrial natriuretic factor

Several forms of the polypeptide atrial natriuretic factor (ANF) have been isolated recently from rat and human atria and identified; they are probably associated with the secretory granules of atrial tissue. The potent ability of ANFs to increase urine sodium content is mediated by their direct action on the kidney. We report here the high intrinsic activity of a synthetic replicate of one form of this molecule, ANF(8-33)(ref. 7), to inhibit directly basal aldosterone secretion and its ability to antagonize the stimulatory effects of adrenocorticotropin (ACTH) and angiotensin II (AN-II) on the secretion of aldosterone by rat adrenoglomerulosa cells in vitro. Our results suggest that ANF is of clinical importance in the management of aldosterone-dependent hypertension by modifying the adrenocortical response to endogenous ACTH and AN-II.     

Purification, sequencing and synthesis of natriuretic and vasoactive rat atrial peptide

Mammalian atria contain potent natriuretic and diuretic substances which exist in high- and low-molecular-weight forms and which appear to be associated with atrium-specific granules. The natriuretic effect of atrial extract is largely accountable for by its renal haemodynamic effects; atrial extracts also antagonize hormone- and non-hormone-induced contraction of the isolated rabbit aorta and isolated rat kidney vasculature. We have completely purified a low-molecular-weight natriuretic and vasoactive substance from rat atria and characterized it as a 24-amino acid peptide. Synthetic peptide, produced by solid-phase synthesis, mimics biological effects of crude atrial extract and purified peptide; its activity is enhanced by slow oxidation, suggesting a disulphide (Cys 4-Cys 20) configuration for the native peptide. If secreted into blood, this atrial natriuretic peptide (' auriculin B') could be a novel peptide hormone of considerable importance to renal and cardiovascular homeostasis.     

运动对心钠素及其基因表达影响的研究

心钠素(Atrial Natriuretic Factor,ANF)是心肌细胞产生和分泌的一种循环激素,具有强大的利钠、利尿、舒张血管及对抗肾素血管紧张素系统等作用.运动对心钠素及其基因表达的影响主要与运动强度、运动时间和运动方式等因素有关,此外缺氧和高应激因素也会影响心钠素及其基因表达.     

运动对心钠素及其基因表达影响的研究

心钠素(Atrial Natriuretic Factor,ANF)是心肌细胞产生和分泌的一种循环激素,具有强大的利钠、利尿、舒张血管及对抗肾素血管紧张素系统等作用。运动对心钠素及其基因表达的影响主要与运动强度、运动时间和运动方式等因素有关,此外缺氧和高应激因素也会影响心钠素及其基因表达。