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 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.     

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.     

Growth restoration of insulin-deficient diabetic rats by recombinant human insulin-like growth factor I

Insulin-like growth factor I (IGF-I) and insulin stem from a common precursor, are structural homologues, act through similar receptors and elicit insulin-like and growth-promoting effects in vitro and in vivo. Serum IGF-I levels are controlled by growth hormone, insulin and nutrition. Insulin-deficient growth-arrested diabetic animals have reduced serum IGF-I levels which are restored towards normal by insulin but not by growth-hormone treatment. Here we show that normal growth of diabetic rate is restored by infusion of recombinant human (rh)IGF-I without normalization of the blood sugar level and that insulin acts via an increase of IGF-I synthesis on growth of diabetic rats. We describe a new mechanism of endocrine control of growth in which IGF-I is the major stimulator at the cellular level. Growth hormone and insulin act mainly by modulating the hepatic synthesis of IGF-I.     

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 .     

人胰岛素样生长因子-Ⅱ基因cDNA克隆与序列分析

本研究通过RT-PCR技术从人胎盘组织中扩增出长为841bp的胰岛素样生长因子-ⅡcDNA,PCR产物经凝胶回收纯化后,克隆在PGEM-T载体的T位点.测序结果和序列分析表明,本人已成功地扩增、克隆了人胰岛素样生长因子-Ⅱ基因cDNA序列.     

Stimulation of tyrosine-specific phosphorylation in vitro by insulin-like growth factor I

Several mitogens elicit tyrosine-specific protein kinase activities. Although the physiological significance of this is unclear, the generality of these reactions implies that this may be an inherent feature of growth factor-growth factor receptor interactions. The observed mitogenic properties of the polypeptide insulin-like growth factor I (IGF-I) indicated that it might also stimulate such activity. We report here that IGF-I stimulates a tyrosine-specific protein kinase in a time- and dose-dependent fashion. The close correspondence between an approximate 50% effective dose (ED50) of phosphorylation and an approximate Kd for IGF-I binding leads us to conclude that a high-affinity IGF-I receptor, not the structurally similar insulin receptor, is the mediator of IGF-I stimulated kinase activity. Immunoprecipitation indicates that both the beta-subunit of the IGF-I receptor and the beta-subunit of the insulin receptor are targets for the IGF-I-stimulated protein kinase.     

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 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.     

Molecular cloning of cDNA encoding a murine haematopoietic growth regulator, granulocyte-macrophage colony stimulating factor

DNA clones specifying the murine granulocyte-macrophage colony stimulating factor have been isolated. This haematopoietic growth factor is encoded by a unique gene specifying a messenger RNA of 1,200 nucleotides and a polypeptide of 118 amino acids. It bears no structural similarity to the functionally related factor, interleukin-3, described recently.     

Localization of insulin-like growth factor genes to human chromosomes 11 and 12

The insulin-like growth factors IGF-I and IGF-II are required for growth and development. Both are single-chain proteins (of 70 and 67 amino acids respectively) derived from precursors by proteolytic processing. IGF-I may be particularly important in promoting normal stature and IGF-II may be a fetal growth hormone. The IGF proteins are probably synthesized by many normal tissues and by some tumours. The secretion of growth factors by tumours and tumour-derived cell lines suggests that they may act as autocrine regulators of cell proliferation. Because of the possible role of these proteins in growth disorders and cancer, and their sequence homology with insulin, we have determined their chromosomal localization. Using somatic cell hybrids and cloned cDNA probes for these proteins, we have assigned the genes for IGF-I and IGF-II to human chromosomes 12 and 11, respectively. We present evidence that the IGF-II gene is located on the short arm of chromosome 11 with a ras proto-oncogene and the insulin structural gene, and also suggest the existence of a fragment length polymorphism using the IGF-I probe.     

Human chromosomal mapping of genes for insulin-like growth factors I and II and epidermal growth factor

Many of the actions previously attributed to pituitary-derived growth hormone are mediated by polypeptide growth factors. These include the insulin-like growth factors I and II (IGF-I and IGF-II), which are members of the insulin family of proteins. We report here the chromosomal mapping of the human genes for IGF-I and IGF-II. IGF-II maps to the short arm of chromosome 11, which also contains the gene for insulin and the proto-oncogene c-Ha-ras1 (ref. 9). IGF-I maps to chromosome 12, which is evolutionarily related to chromosome 11 and carries the gene for the proto-oncogene c-Ki-ras2 (refs 10,44). We have also localized the human gene for an unrelated polypeptide hormone, epidermal growth factor, to chromosome 4q, in the same region as another specialized growth factor, T-cell growth factor. We speculate that these map assignments reflect the existence of gene families involved in growth control.     

Computer-based characterization of epidermal growth factor precursor

The cDNA sequence of the precursor of mouse epidermal growth factor (EGFP) has recently been reported by two groups, both of whom noted the presence of repeated similar segments, each about 40 residues long. One of these repeat units overlaps with the sequence of epidermal growth factor itself. The sequence of epidermal growth factor has been reported to be similar to that of pancreatic secretory trypsin inhibitor (PSTI) and a somewhat better match has been found with part of the sequence of bovine factor X, one of the blood coagulating factors. We report here that there is an even stronger similarity between the sequences of some of the repeat units of epidermal growth factor precursor and certain segments in factor X. This sequence similarity is also apparent in comparisons with other blood coagulation factors. On the basis of these sequence comparisons we suggest a scheme for the evolution of the epidermal growth factor precursor. We have also identified certain structural features in the precursor sequence that bear on the way in which the mature epidermal growth factor is generated.     

Structure of rat atrial natriuretic factor precursor deduced from cDNA sequence

The atrium of the heart contains peptides, termed atrial natriuretic factors ( ANFs ), diuretic and smooth-muscle-relaxing activities. In view of its potent effects on salt metabolism in the kidney and on vascular smooth muscle, ANF is considered to play an important part in the control of fluid volume and vascular function. Several different ANF peptides varying in size have been isolated and their amino acid sequences determined. Analysis of the sequences of the peptides suggests that they are derived by proteolysis from the same precursor. To examine this hypothesis, we have cloned cDNAs of the ANF precursor using rat atrial mRNA, determined its nucleotide sequence and deduced its amino acid sequence. The ANF precursor consists of 152 amino acid residues including a putative signal peptide sequence. This sequence contains the amino acid sequences of all the ANF peptides reported to date.     

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.     

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.     

肝癌胰岛素样生长因子II基因启动子P3甲基化的检测及意义

目的:建立检测肝癌胰岛素样生长因子II(IGF-II)基因启动子P3甲基化状态的方法.方法:培养三株肝癌细胞系(BEL7402、SMMC7721、HepG2),选取正常肝组织3例;提取基因组DNA,亚硫酸氢钠修饰,巢式聚合酶链反应(nested PCR)加甲基化特异性PCR(MSP)检测IGF-II基因启动子P3的甲基化状态,PCR产物经克隆、测序验证.结果:三株肝癌细胞系IGF-II基因启动子P3去甲基化,正常肝组织IGF-II基因启动子P3甲基化,目的片段无突变,甲基化修饰完全.结论:巢式PCR加甲基化特异性PCR可准确检测IGF-II基因启动子P3的甲基化状态.     

肝癌胰岛素样生长因子Ⅱ基因启动子P3甲基化的检测及意义

目的：建立检测肝癌胰岛素样生长因子Ⅱ(IGF-Ⅱ)基因启动子P3甲基化状态的方法。方法：培养三株肝癌细胞系(BEL7402、SMMC7721、HepG2)，选取正常肝组织3例；提取基因组DNA，亚硫酸氢钠修饰，巢式聚合酶链反应(nested PCR)加甲基化特异性PCR(MSP)检测IGF-Ⅱ基因启动子P3的甲基化状态，PCR产物经克隆、测序验证。结果：三株肝癌细胞系IGF-Ⅱ基因启动子P3去甲基化，正常肝组织IGF-Ⅱ基因启动子P3甲基化，目的片段无突变，甲基化修饰完全。结论：巢式PCR加甲基化特异性PCR可准确检测IGF-Ⅱ基因启动子P3的甲基化状态。