A novel peptide which stimulates adenylate cyclase: molecular cloning and characterization of the ovine and human cDNAs

A novel neuropeptide which remarkably stimulates adenylate cyclase in rat anterior pituitary cell cultures has been recently isolated from ovine hypothalami by A. Arimura and his collaborators (Biochem.Biophys.Res.Commun.164, 567-574(1989)). This peptide was designated as PACAP38(Pituitary Adenylate Cyclase Activating Polypeptide with 38 residues). In an attempt to investigate physiological implications of PACAP38, we have succeeded in cloning the cDNAs encoding the precursor of PACAP38 from ovine hypothalamus and human testis. An ovine cDNA encodes a protein of 176 amino acids in which PACAP38 is proceeded by a putative signal peptide and a "pro"-region (107 amino acids), and followed by a Gly-Arg-Arg sequence for proteolytic processing and amidation. Deduced amino-acid sequence of human PACAP38 was completely identical to that of the ovine isolated peptide. Cloning of PACAP38 cDNAs confirms the expression of the corresponding mRNAs and the presence of this neuropeptide in ovine hypothalamus and also in human testis.     

Cloning and sequence analysis of cDNA for rat corticotropin-releasing factor precursor

DNA complementary to the rat hypothalamic mRNA coding for the corticotropin-releasing factor precursor (prepro-CRF) has been cloned by screening a cDNA library with a human genomic DNA probe. Nucleotide sequence analysis of the cloned cDNA has revealed that rat prepro-CRF consists of 187 amino acid residues including a putative signal peptide. The CRF and putative signal peptide regions are more highly conserved among rat, human and ovine prepro-CRF than is the cryptic portion.     

Molecular cloning of cDNA for the import precursor of human coupling factor 6 of H(+)-ATP synthase in mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat factor 6 as a probe. The sequence was composed of 466 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions on the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame were found to consist of 108 and 76 amino acid residues with molecular weights of 12,596 and 8,969, respectively. The presequence of 32 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

Complete nucleotide sequence of ovine alpha-lactalbumin mRNA

The nucleotide sequence of ovine alpha-lactalbumin mRNA has been determined by chemical sequencing of two cDNA recombinant plasmids and a primer extension product. Ovine alpha-lactalbumin mRNA contains 723 nucleotides (excluding the poly(A) tail), with a 5' non-coding region of 26 nucleotides, followed by the 426 nucleotides of the coding region which determines a sequence signal of 19 amino acid residues and the 123 amino acid residues of mature alpha-lactalbumin. The coding region is followed by a 3' untranslated sequence of 271 nucleotides. The derived amino acid sequence of ovine pre-alpha-lactalbumin differs from that of its bovine counterpart by 8 amino acid substitutions, all but one originating from single mutations. Comparison of sequences of guinea pig, rat and human alpha-lactalbumin mRNAs with their ovine and bovine counterparts has revealed that these molecules have rapidly evolved. The highest degree of conservation was observed in the region coding for the mature protein and corresponds essentially to sequences which interact with UDP-galactosyltransferase and Ca2+ ions.     

Molecular cloning and nucleotide sequence of cDNA coding for rat brain cholecystokinin precursor

A mixture of 14-mer oligodeoxynucleotides was used for the screening of a cDNA clone coding for a cholecystokinin (CCK) precursor from a cDNA library for rat brain microsomal poly(A)RNA. The longest insert is 718 bp long which was verified to contain a nearly full-length cDNA sequence coding for rat CCK precursor, because the size of CCK mRNA was estimated to be about 850 bases long by Northern blotting analysis. Sequence analysis revealed 110 bp in the 5'-untranslated region, 345 bp in the amino acid coding region corresponding to the CCK precursor and 263 bp in the 3'-noncoding region which contains polyadenylation signal AUUAAA and the poly(A) sequence. The precursor may contain a 28 amino acid signal peptide and 12 additional amino acids at the carboxyl terminus.     

Chemical characterization of vasoactive intestinal polypeptide-like immunoreactivity in ovine hypothalamus and intestine.

Two forms of pituitary adenylate cyclase activating polypeptides with 38 (PACAP38) and 27 residues (PACAP27) respectively were recently isolated from ovine hypothalamic tissues. The N-terminal 28 amino acids sequence of PACAP was found to have 68% homology with porcine vasoactive intestinal peptide (VIP). In order to determine whether the primary structure of VIP of ovine hypothalamus is identical with porcine VIP or similar to PACAP, VIP immunoreactivity as determined by radioimmunoassay for porcine VIP was isolated in a pure form from ovine hypothalamic extracts. VIP was also isolated from ovine intestine. Amino acid analysis as well as amino acid sequence analysis showed that ovine hypothalamic and intestinal VIP were identical to porcine VIP, but different from PACAP.     

Molecular cloning of cDNA for the import precursor of human subunit B of H(+)-ATP synthase in mitochondria.

The nucleotide sequence of the import precursor of subunit b of human H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a human kidney cDNA library with a cDNA for rat subunit b as a probe. The sequence was composed of 1,134 nucleotides including a coding region for the import precursor of subunit b and noncoding regions on the 5'- and 3'-sides. The import precursor of subunit b and its mature polypeptide deduced from the open reading frame were found to consist of 256 and 214 amino acid residues with molecular weights of 28,893 and 24,610, respectively. The presequence of 42 amino acids could be the import signal peptide for directing the protein into the mitochondrial matrix.     

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.     

Nucleotide sequence of the cDNA encoding the precursor for mitochondrial serine:pyruvate aminotransferase of rat liver

The nucleotide sequence of the mRNA coding for the precursor of mitochondrial serine:pyruvate aminotransferase of rat liver was determined from those of cDNA clones. The mRNA comprises at least 1533 nucleotides, except the poly(A) tail, and encodes a polypeptide consisting of 414 amino acid residues with a molecular mass of 45,834 Da. Comparison of the N-terminal amino acid sequence of mitochondrial serine:pyruvate aminotransferase with the nucleotide sequence of the mRNA showed that the mature form of the mitochondrial enzyme consisted of 390 amino acid residues of 43,210 Da. The amino acid composition of mitochondrial serine:pyruvate aminotransferase deduced from the nucleotide sequence of the cDNA showed good agreement with the composition determined on acid hydrolysis of the purified protein. The extra 24 amino acid residues correspond to the N-terminal extension peptide (pre-sequence) that is indispensable for the specific import of the precursor protein into mitochondria. In the extension peptide there are four basic amino acids distributed among hydrophobic amino acids and, as revealed on helical wheel analysis, the putative alpha-helical structure of the peptide was amphiphilic in nature. The secondary structures of the mature serine:pyruvate aminotransferase and three other aminotransferases of rat liver were predicted from their amino acid sequences. Their secondary structures exhibited a common feature and so we propose the specific lysine residue which binds pyridoxal phosphate as the active site of serine:pyruvate aminotransferase.     

Expression of kappa-casein in normal and neoplastic rat mammary gland is under the control of prolactin

An 820-nucleotide-long cDNA clone for the kappa-casein (the casein micelle-stabilizing protein) from rat mammary gland was isolated, and its nucleotide sequence was determined. The deduced amino acid sequence from the nucleotide sequence revealed a signal peptide, 21 amino acids long, and a mature protein of 157 amino acids. The signal peptide of rat kappa-casein was highly homologous to that of the precursor to ovine kappa-casein. However, little homology was apparent when the mature kappa-casein protein sequences from ovine or bovine sources were compared with rat kappa-casein. The kappa-casein mRNA content of the mammary tissue was found to increase during its functional differentiation. Prolactin appears to modulate the production of kappa-casein mRNA. Mammary glands of virgin females had no detectable kappa-casein mRNA; however, a marked induction of kappa-casein mRNA was obtained by intravenous infusion of prolactin. Mammary carcinomas did not follow the same pattern. 7,12-Dimethylbenz[a]anthracene-induced mammary carcinomas had normally low levels of kappa-casein mRNA, but intravenous prolactin infusion increased the levels by 2-fold. The MTW9 mammary carcinoma that grows only in the presence of high levels of mammotropic hormones had kappa-casein mRNA content equivalent to that in 10-day lactating rat mammary gland. Continuous venous infusion of prolactin to MTW9 mammary carcinoma did not modify the kappa-casein mRNA levels. Nitrosomethylurea-induced mammary carcinomas had no detectable kappa-casein mRNA, and intravenous prolactin infusion was unable to induce it.     

Rat apolipoprotein C-II lacks the conserved site for proteolytic cleavage of the pro-form.

Apolipoprotein C-II (apoC-II) plays a critical role in the metabolism of plasma lipoproteins as an activator for lipoprotein lipase. Human apoC-II consists of 79 amino acid residues (pro-apoC-II). A minor fraction is converted to a mature form by cleavage at the site QQDE releasing the 6 amino-terminal residues. We have cloned and sequenced the cDNA for rat apoC-II from a liver cDNA library using human apoC-II cDNA as a probe. The cDNA encodes a protein of 97 amino acid residues including a signal peptide of 22 amino acid residues. There is approximately 60% similarity between the deduced amino acid sequence of rat apoC-II and other apoC-II sequences presently known (human, monkey, dog, cow, and guinea pig). Compared to these, rat apoC-II is one residue shorter at the carboxyl terminus. Furthermore, there is a deletion of 3 amino acid residues (PQQ) in the highly conserved cleavage site where processing from pro- to mature apoC-II occurs in other species. Accordingly, rat apoC-II isolated from plasma was mainly in the pro-form. Northern blot analyses indicated that rat apoC-II is expressed both in liver and in small intestine.     

Cloning and characterization of the growth hormone-dependent insulin-like growth factor binding protein (IGFBP-3) in the rat.

We report for the first time the complete amino acid sequence for the growth hormone dependent insulin-like growth factor binding protein (IGFBP-3) in the rat. A human IGFBP-3 clone was generated using the polymerase chain reaction (PCR) and used to screen a rat liver cDNA library. cDNA clones of the rat IGFBP-3 were isolated and the full amino acid sequence deduced. The sequence begins with a putative, 26 amino acid signal peptide followed by a 265 amino acid binding protein. The amino acid sequence is over 80% homologous with the equivalent human IGFBP-3 form and shows complete conservation of 18 cysteine residues that are clustered at the amino and carboxy ends of the protein. IGFBP-3 is the binding subunit of the major circulating IGFBP in the rat, and hence the availability of precise structural data and cDNA probes provides an important opportunity for a detailed study of the control of IGFBP-3 synthesis at the level of gene expression.     

Primary structure of rat liver alkaline phosphatase deduced from its cDNA.

Rat liver alkaline phosphatase (ALP) was markedly induced by treatment of rats by bile-duct ligation and colchicine injection. Taking this advantage for enrichment of ALP mRNA, we constructed a lambda gt11 liver cDNA library using polyadenylated RNA prepared from the treated rat liver, and isolated an ALP cDNA clone. The 2165 bp cDNA contained an open reading frame that encodes a 524-amino-acid-residue polypeptide with a predicted molecular mass of 57737 Da. The precursor protein contained a presumed signal peptide of 17 amino acid residues followed by 28 amino acid residues identical with the N-terminal sequence determined from the purified rat liver ALP. It was also confirmed that amino acid sequences of two CNBr-cleavage peptides obtained from liver ALP were contained within the cDNA-encoded protein. Five possible N-linked glycosylation sites were found in the molecule and a highly hydrophobic amino acid sequence at the C-terminus. The deduced polypeptide of rat liver ALP showed 88% homology to that of the human liver-type enzyme in osteosarcoma cells. RNA blot hybridization analysis identified a single species of ALP mRNA with 2.7 kb in both the control and the treated rat livers. An approx. 20-fold increase of the mRNA was detected in the treated liver at 12 h after the onset of stimulation, compared with that in the control liver.     

Chicken apolipoprotein A-I: cDNA sequence,tissue expression and evolution

Using an antibody against chicken apolipoprotein (apo) A-I, we identified multiple cDNA clones for the protein in two intestinal cDNA libraries in λgtll. The complete nucleotide sequence of chicken apoA-I cDNA was determined. The sequence predicts a mature protein of 240 amino acids, a 6-amino acid propeptide and an 18-amino acid signal peptide. Using a ³²P-cDNA probe, we detected the presence of apoA-I mRNA in 21 day old chicken intestine, liver, kidney, spleen, breast muscle and brain. The primary sequence of apoA-I contains numerous tandem repeats of 11 and 22 residues in a manner similar to the mammalian proteins. Our analysis of apoA-I sequences from human, rabbit, dog, rat, and chicken indicates that the rate of amino acid substitution is considerably faster in the rat lineage than in other mammalian lineages.     

cDNA cloning and sequencing for the import precursor of coupling factor 6 in H(+)-ATP synthase from rat liver mitochondria

The nucleotide sequence of the import precursor of coupling factor 6 (factor 6) of rat liver H(+)-ATP synthase has been determined from a recombinant cDNA clone isolated by screening a rat liver cDNA library with a probe DNA. The sequence was composed of 458 nucleotides including a coding region for the import precursor of factor 6 and noncoding regions of both the 5'- and 3'-sides. The import precursor of factor 6 and its mature polypeptide deduced from the open reading frame consisted of 108 and 76 amino acid residues with a molecular weight of 12,494 and 8,927, respectively. The presequence of 32 amino acids could be the import signal peptide which serves to direct the protein into the mitochondrial matrix.     

Isolation and sequence analysis of a cDNA encoding rat liver alpha-L-fucosidase.

cDNA clones for alpha-L-fucosidase were isolated from a rat liver lambda gt11 expression library by using both monospecific polyclonal antibodies against the affinity-purified enzyme and biotinylated rat liver fucosidase cDNA sequences as probes. The largest clone, lambda FC9, contained a 1522 bp full-length cDNA insert (FC9) that encoded the 434-amino acid-residue subunit (Mr 50439) of rat liver alpha-L-fucosidase. A putative signal peptide 28 amino acid residues in length preceded the sequence for the mature protein. In addition, FC9 specified for 11 nucleotide residues of 5' untranslated sequence, 78 nucleotide residues of 3' untranslated sequence and a poly(A) tail. The deduced amino acid sequence from FC9 in conjunction with the experimentally determined N-terminus of the mature enzyme suggested that rat liver fucosidase did not contain a pro-segment. However, there was the possibility of limited N-terminal processing (one to five amino acid residues) having occurred after removal of the predicted signal peptide. Amino acid sequences deduced from FC9 were co-linear with amino acid sequences measured at the N-terminus of purified fucosidase and on two of its CNBr-cleavage peptides. An unusual aspect of rat liver alpha-L-fucosidase protein structure obtained from the FC9 data was its high content of tryptophan (6%). The coding sequence from FC9 showed 82% sequence identity with that from a previously reported incomplete human fucosidase sequence [O'Brien, Willems, Fukushima, de Wet, Darby, DiCioccio, Fowler & Shows, (1987) Enzyme 38, 45-53].     

Molecular cloning and sequencing of a rat preprogastrin complementary deoxyribonucleic acid

Gastrin biosynthesis involves a complex series of posttranslational modifications; their elucidation requires a knowledge of the structure of the gastrin precursor. The complete structure of rat preprogastrin was deduced from the nucleotide sequence of a full length cDNA clone isolated from a rat antral cDNA library. Northern blot hybridization analysis of rat antral RNA together with human antral RNA, reveals a single mRNA species of approximately 670 bases. Comparison of this sequence with those of porcine and human gastrin reveals extensive (73%) homology in the gastrin coding region as well as short regions of conserved nucleotides in the noncoding regions. The rat sequence encodes a preprogastrin of 104 amino acids which consists of a signal peptide, a 37 amino acid prosegment; and the gastrin 34 sequence, followed by a glycine (the amide donor), and flanked by pairs of arginine residues. Cleavage at an internal pair of lysine residues yields gastrin 17. Unlike the human and porcine sequences, rat preprogastrin contains a 9 amino acid carboxy-terminal extension peptide (-Ser-Ala-Glu-Glu-Glu-Asp-Gln-Tyr-Asn) which is homologous to the midportion of gastrin 17 including the site of tyrosine sulfation.     

Cloning, sequence analysis and expression of a cDNA encoding a novel insulin-like growth factor binding protein (IGFBP-2).

Insulin-like growth factors bind with high affinity to specific binding proteins in extracellular fluids. To identify structural characteristics of IGF-binding proteins that might define their physiological roles, we determined the complete primary structure of a novel human IGF-binding protein (IGFBP-2) from a cloned cDNA. The cDNA encodes a 328 amino acid IGF-binding protein precursor which contains a 39-residue signal peptide. The mature 289 amino acid IGFBP-2 has a predicted Mr of 31,325. Chinese hamster ovary (CHO) cells stably transformed with the IGFBP-2 cDNA secreted a 36 kd protein which bound, with different affinities, IGFII and IGFI, but did not bind insulin. The predicted protein sequence of this IGF-binding protein shares extensive amino acid homology (greater than 85%) with the IGF-binding protein secreted by rat BRL-3A cells, but less than 40% homology with human IGFBP-1. Therefore IGFBP-2, and not IGFBP-1 as previously suggested, represents the human homologue of the rat BRL-BP (alpha IGFBP-2). Moreover, from alignment of the predicted protein sequences of IGFBP-1 and IGFBP-2, extensive conservation of the distribution of cysteine residues is observed. Although the overall amino acid homology shared by these proteins is not high, we suggest that they represent a family of structurally related human IGFBPs. Southern blot analysis of human DNA demonstrates that IGFBP-2 is encoded by a single-copy gene, different from that of IGFBP-1.