Cloning and expression of a complementary DNA encoding a bovine adrenal angiotensin II type-1 receptor

Angiotensin II elicits different responses which affect cardiovascular, neuronal and electrolyte transport regulation. To understand the mechanisms responsible for its various actions, the receptor for angiotensin II has long been sought, but numerous attempts to purify the receptor have been unsuccessful owing to its instability and low concentration. We report here the expression cloning of a complementary DNA encoding a bovine angiotensin II receptor to overcome these difficulties. The receptor cDNA encodes a protein of 359 amino-acid residues with a transmembrane topology similar to that of other G protein-coupled receptors. COS-7 cells transfected with the cDNA expressed specific and high-affinity binding sites for angiotensin II, angiotensin II antagonist and a non-peptide specific antagonist for type-1 receptor. Dithiothreitol inhibited ligand binding. The concentration of intracellular Ca2+ and of inositol-1,4,5-trisphosphate increased in the transfected COS-7 cells in response to angiotensin II or angiotensin III, indicating that this receptor is the type-1 receptor for angiotensin II. Northern blot analysis revealed that the messenger RNA for this receptor is expressed in bovine adrenal medulla, cortex and kidney.     

Cloning of a cDNA encoding a non-isopeptide-selective subtype of the endothelin receptor

Endothelin-1 was initially identified as a 21-residue potent vasoconstrictor peptide produced by vascular endothelial cells, but was subsequently found to have many effects on both vascular and non-vascular tissues. The discovery of three isopeptides of the endothelin family, ET-1, ET-2 and ET-3, each possessing a diverse set of pharmacological activities of different potency, suggested the existence of several different endothelin receptor subtypes. Endothelins may elicit biological responses by various signal-transduction mechanisms, including the G protein-coupled activation of phospholipase C and the activation of voltage-dependent Ca2+ channels. Thus, different subtypes of the endothelin receptor may use different signal-transduction mechanisms. Here we report the cloning of a complementary DNA encoding one subtype belonging to the superfamily of G protein-coupled receptors. COS-7 cells transfected with the cDNA express specific and high-affinity binding sites for endothelins, responding to binding by the production of inositol phosphates and a transient increase in the concentration of intracellular free Ca2+. The three endothelin isopeptides are roughly equipotent in displacing 125I-labelled ET-1 binding and causing Ca2+ mobilization. A messenger RNA corresponding to the cDNA is detected in many rat tissues including the brain, kidney and lung but not in vascular smooth muscle cells. These results indicate that this cDNA encodes a 'nonselective' subtype of the receptor which is different from the vascular smooth muscle receptor.     

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.     

Cloning and expression of a cDNA encoding an endothelin receptor

Endothelins are a newly described peptide family consisting of three peptides (ET-1, ET-2 and ET-3) which are the most potent vasoconstrictive peptides known. They are crucial in the regulation of vascular smooth muscle tone. The diverse functions of endothelins are thought to be mediated by interaction with many different receptors coupled to the inositol phosphate/calcium ion messenger pathway. However, because of the structural resemblance of the three peptides, the presence and nature of multiple endothelin receptors remain to be elucidated. We report here the cloning of a complementary DNA encoding a bovine endothelin receptor, which has a transmembrane topology similar to that of other G protein-coupled receptors and shows specific binding, with the highest selectivity to ET-1 in animal cells transfected with the cloned cDNA. This receptor messenger RNA is widely distributed in the central nervous system and peripheral tissues, particularly in the heart and lung. Our results support the view that there are other receptor subtypes.     

Isolation of cDNA clones encoding T cell-specific membrane-associated proteins

Of 10 distinct cloned DNA copies of mRNAs expressed in T lymphocytes but not in B lymphocytes and associated with membrane-bound polysomes, one hybridizes to a region of the genome that has rearranged in a T-cell lymphoma and several T-cell hybridomas. These characteristics suggest that it encodes one chain of the elusive antigen receptor on the surface of T lymphocytes.     

Sequence of a cDNA clone encoding human preproinsulin-like growth factor II

The insulin-like growth factors (IGF) I and II are single-chain serum proteins of 70 and 67 amino acids, respectively, which are synthesized by the liver and possibly other tissues. They are probably required for normal fetal and postnatal growth and development. They also stimulate the growth of cultured cells, possibly by controlling the progression through the G1 phase of the cell cycle. In contrast to IGF-II whose concentration does not vary during postnatal development, the serum levels of IGF-I increase several-fold to adult levels during puberty. The serum concentration of IGF-I is a sensitive monitor of growth hormone levels and is decreased in individuals with growth hormone deficiency and elevated in those with growth hormone-secreting tumours. As a first step in studying the biosynthesis of these proteins and elucidating their role(s) in normal development and in tumorigenesis, we have isolated and sequenced cDNAs prepared from adult human liver mRNA which encode the precursors to IGF-I and -II. We report here the sequence of a cDNA encoding a 180-amino acid protein which is the precursor to IGF-II.     

Isolation and characterization of a cDNA clone encoding the murine homologue of the human 20K T3/T-cell receptor glycoprotein

The antigen receptor on the surface of human T lymphocytes, which consists of a heterodimer of relative molecular mass (Mr) 90,000 (90K) (alpha- and beta-chains), is associated with the T3 antigen (gamma = 25K, delta = 20K and epsilon = 20K). A working model for the mode of action of the T3/T-cell receptor complex is that the clonotypic alpha- and beta-chains are involved in the recognition and binding of antigen in the context of polymorphic major histocompatibility complex (MHC) gene products on the surface of target cells. Antigen binding by the clonotypic receptor probably results in conformational changes in this structure which are recognized by and subsequently trigger the associated T3 complex to transmit signals into the cell, resulting in a proliferative response. The similarity in structure between murine and human clonotypic antigen receptors suggests that such a mechanism of recognition and activation also exists in mouse T lymphocytes, but so far there has been no evidence for the existence of a murine T3 complex. Here we demonstrate the existence of a T3 delta-chain mRNA in murine T lymphocytes. Our sequence data strongly suggest that this mouse mRNA codes for a complete T3 delta polypeptide chain and reveal some interesting properties of the protein.     

Molecular cloning of cDNA encoding human interleukin-2 receptor

The human interleukin-2 (IL-2) receptor was purified by affinity chromatography using the anti-Tac monoclonal antibody, and its N-terminal amino acid sequence was determined. Complementary DNA clones were isolated and sequenced to reveal the primary structure of the IL-2 receptor precursor, which has 272 amino acid residues. The receptor is separated into two domains by a putative 19-residue transmembrane region. Two mRNAs (1.4 and 3.5 kilobases) hybridizing to the cDNA clone were found in human T cells bearing the IL-2 receptor. The cDNA directed synthesis of the IL-2 receptor in COS cells.     

Isolation of cDNA clones encoding the 20K non-glycosylated polypeptide chain of the human T-cell receptor/T3 complex

The antigen receptor on human T lymphocytes consists of two variable immunoglobulin-like glycoproteins, alpha and beta, which occur in association with three invariable T3 membrane proteins. In humans two of these proteins, T3-gamma and T3-delta, are glycoproteins of relative molecular mass (Mr) 25,000 (25K) and 20,000 (20K), respectively, while the third, T3-epsilon, is a 20K non-glycosylated protein. On the surface of murine T cells, a non-glycosylated protein dimer composed of 17K subunits (T3-zeta) is found associated with the T-cell receptor alpha and beta chains and the three T3-like polypeptide chains. It is generally accepted that major histocompatibility complex-restricted antigen recognition is a function of the alpha-beta heterodimer. This has led to the postulation that the proteins of the T3 complex are involved in the signal transduction that immediately follows antigen recognition via the antigen receptor. Events believed to be involved in early T-cell activation, such as rapid increases in phosphatidylinositol turnover and free intracellular calcium, can be triggered by antibodies directed against either the T3 complex or the clonotypic receptor. We have previously reported our findings on the cloning of the complementary DNA and genomic structure encoding both the human and murine 20K glycoprotein, T3-delta (refs 11-13). We now present our results on the cloning of the cDNA encoding the human 20K non-glycosylated chain, T3-epsilon.     

Isolation and cloning of a novel cDNA LDBl encoding human LIM domain binding protein

Primers for screening cDNA library have been designed according to EST AA453734 which is corresponding to the mouse LIM domain binding protein Ldb1. Arrayed human fetal brain cDNA library has been screened by PCR and routine hybridization method. A 2398 bp-cD-NA clone has been obtained. The cDNA encodes a 347 amino acids protein highly homologous to the mouse Ldb1, Xenopus Xldb1 and Drosophila Chip. It also contains an LIM binding domain and a nuclear localization signal. It has been named LDB1 (LIM domain binding protein 1), GenBank accession number is AF052389. Northern blot showed a 2.4 kb band, and the expression amounts of LDB1 in heart, brain and lung were considerably higher than those in other tissues.     

Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit

We have isolated a complementary DNA clone containing sequences homologous to those encoding the alpha-subunit of a mouse muscle nicotinic acetylcholine receptor. Based on the structural similarities between the encoded protein and the muscle acetylcholine receptor alpha-subunit, and the presence of hybridizing RNA species in the brain, we propose that this clone codes for a neural nicotinic acetylcholine receptor alpha-subunit.     

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.     

人EGF受体胞外域cDNA克隆及其序列分析

目的：克隆编码人表皮生长因子受体(EGFR)胞外域的cDNA,构建可溶性EGFR(sEGFR)的哺乳动物细胞表达载体。方法：以RT—PCR方法从人胎盘中克隆编码EGFR胞外域的cDNA,测定其序列,构建sEGFR真核表达载体。结果：从人胎盘绒毛组织中成功克隆编码EGFR胞外域的cNDA,通过PCR方法在起始位点前加入Kozak序列并添加终止密码,构建了sEGFR的真核表达载体。该基因编码的蛋白质包含信号肽以及L1、S1、12和S2等4个结构域。序列分析表明该基因有3个碱基与以往报道的基因不同,即1562G→A、1620G→C、1887T→A,前两个改变导致氨基酸Arg497Lys和Lys516Asn的改变,后一个为同义突变Thr605。结论：成功克隆编码EGFR胞外域的cDNA,并构建了sEGFR真核表达载体。     

人尿激酶原全长cDNA基因的分离与鉴定

将两对人工合成的寡聚核苷酸，经体外延伸后制备成探针。用此探针，从一种以λgt11为载体的人胎盘cDNA库中，经3次纯化杂交筛选，分离出21个阳性克隆。对其中4个阳性克隆进行了限制性内切酶图谱、Southern印迹法及部分序列分析鉴定。证明了四个克隆子中的3个，除具有完整的人尿激酶原cDNA编码区外，还具有包含起密码子在内的20个氨基酸信号肽部分及5′端、3′端非编码区。这些阳性克隆子可用于人尿激酶原的基因工程研究。     

Cloning of cDNA encoding the murine IgG1 induction factor by a novel strategy using SP6 promoter

Complementary DNA encoding the IgG1 induction factor, the first lymphokine directed to B lymphocytes, from a murine T-cell line has been cloned using a new strategy. The putative primary amino-acid sequence was deduced from the nucleotide sequence determined. The lymphokine synthesized by the direction of this cloned cDNA has many other functions, such as production of B-cell growth factor-1 and induction of Ia on B cells.