Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively.     

Molecular cloning of cDNA encoding human prointerleukin-6

The data are presented on the cloning and sequencing of cDNA coding for human interleukin-6. The variability of cDNA proIL-6 cloned from different cellular sources was studied. The variability of cDNA proIL-6 may be expressed as heterogeneity of 5'- and 3'-end sequences of cDNA as well as single base-pair changes.     

Molecular cloning of cDNA encoding porcine interleukin-15

Interleukin-15 (IL-15) is a recently identified growth and differentiation factor with an important potential role in the initial immune responses to infection. To enable the study of the role of this cytokine in the protective immune-mechanisms generated against parasitic diseases of swine, cDNA was generated from a macrophage enriched adherent cell population from peripheral blood mononuclear cells (PBMC). This cDNA was used for the enzymatic amplification of the porcine IL-15 sequence using human IL-15-derived primers. The open-reading frame of the porcine IL-15 cDNA is 486 base pairs (bp) in length and encodes a 162-amino-acid (aa) protein. Comparisons of the predicted swine protein sequence with those predicted from human, bovine and mouse IL-15 sequences indicate similarities of 82.1, 84.6, and 71.6%, respectively.     

Novel bradykinins and their precursor cDNAs from European yellow-bellied toad (Bombina variegata) skin.

Two novel bradykinin-related peptides (Ala3,Thr6)-bradykinin and (Val1,Thr3,Thr6)-bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow-bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK-1 and BVK-2, respectively, were cloned from a skin library by 3'- and 5'-RACE reactions. BVK-1 contained an open-reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)-bradykinin and similarly, the open-reading frame of BVK-2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)-bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.     

Molecular cloning and expression of a cDNA encoding the secretin receptor.

Secretin is a 27 amino acid peptide which stimulates the secretion of bicarbonate, enzymes and potassium ion from the pancreas. A complementary DNA encoding the rat secretin receptor was isolated from a CDM8 expression library of NG108-15 cell line. The secretin receptor expressed in COS cells could specifically bind the iodinated secretin with high and low affinities. Co-expression of the secretin receptor with the alpha-subunit of rat Gs protein increased the concentration of the high affinity receptor in the membrane fraction of the transfected COS cells. Secretin could stimulate accumulation of cAMP in COS cells expressing the cloned secretin receptor. The nucleotide sequence analysis of the cDNA has revealed that the secretin receptor consists of 449 amino acids with a calculated Mr of 48,696. The secretin receptor contains seven putative transmembrane segments, and belongs to a family of the G protein-coupled receptor. However, the amino acid sequence of the secretin receptor has no significant similarity with that of other G protein-coupled receptors. A 2.5 kb mRNA coding for the secretin receptor could be detected in NG108-15 cells, and rat heart, stomach and pancreatic tissue.     

Molecular cloning and characterization of a cDNA encoding proline transporter in rice

A cDNA encoding a proline (Pro) transporter (ProT) was isolated and characterized from a cDNA library prepared from 14-d-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the rice ProT protein (OsProT) had 68.8% homology to the ProT protein 1 from Arabidopsis thaliana and 59.6% homology to that from Lycopersicon esculentum. Northern blot analysis revealed that the gene for OsProT (OsProT) was expressed in all organs examined, comparatively strongly in leaf sheath and stem. Salt treatment did not induce expression of OsProT but strongly induced expression of the gene for delta1-pyrroline-5-carboxylate synthetase (P5CS), a key enzyme in Pro biosynthesis. Southern blot analysis revealed that OsProT has a gene family. OsProT specifically transported L-Pro in a transport assay using Xenopus laevis oocytes.     

Molecular cloning of a full-length cDNA encoding the hemagglutinin-neuraminidase glycoprotein of Sendai virus

We cloned a full-length complementary DNA for the hemagglutinin-neuraminidase (HN) mRNA of Sendai virus (HVJ) using a synthetic 27-mer as a probe. Nucleotide sequence analysis showed that there is a long open reading frame on the mRNA that encodes a protein of 575 amino acids. The deduced amino acid sequence indicated that only one hydrophobic region sufficiently long to anchor the protein in the membrane and located near the N-terminus (amino acids 35-60). It is suggested that HN protein is oriented with its N-terminus inside the membrane.     

A family of bombinin-related peptides from the skin of Bombina variegata.

A peptide fraction was isolated from the skin of Bombina variegata that showed antimicrobial activity. This fraction contained several molecular species, all of them consisting of 27 amino acid residues, with a constant C-terminal region (from residues 14-27), including an amidated carboxyl end and a variable N-terminal segment. These peptides are related but not identical to bombinin [Csordas, A. & Michl, H. (1970) Monatsh. Chem. 101, 182-189]. By using synthetic oligonucleotides corresponding to the C-terminal region of the peptides, a cDNA library from the skin of B. variegata was screened and several positive clones coding for the corresponding peptide precursors were isolated and sequenced. Each clone contained the genetic information for a different bombinin-like peptide. The antimicrobial activity towards different bacterial species of a synthetic peptide corresponding to one of the variants deduced from cDNA sequences was tested. This peptide was found to be mainly active against different isolates of Staphylococci and Escherichia coli.     

野桑蚕酚氧化酶原基因cDNA的分子克隆及其特征

酚氧化酶在昆虫的免疫防御机制中起着重要作用。利用RT-PCR和RACE方法,克隆了野桑蚕酚氧化酶原基因,获得了其cDNA序列。该序列长2 134 bp,含有一个2 082 bp的完整开放阅读框,编码一个由693个氨基酸残基组成的蛋白质。推导的氨基酸序列与其他鳞翅目昆虫PPO2基因相应氨基酸序列有较高的同源性,该序列具有它们的PPO基因所共有的典型特征。组织特异性表达分析表明了该基因在野桑蚕5龄幼虫的血细胞、体壁、头部、精巢、卵巢、脂肪体和中肠等组织及其不同的发育阶段均有表达。这些结果为进一步研究野桑蚕酚氧化酶原基因的功能提供了分子基础。     

Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-λgt11 expression library using antibodies to human sperm surface antigens belonging to 14–18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46–54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH₂-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1λT vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14–18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism. Mol. Reprod. Dev. 48:449–457, 1997. © 1997 Wiley-Liss, Inc.     

Molecular cloning and expression of a mouse muscle cDNA encoding adenylosuccinate synthetase

Adenylosuccinate synthetase (EC 6.3.4.4) catalyzes the first step in formation of AMP from IMP. At least two isozymes exist in vertebrate tissue. An acidic form, present in most tissues, has been suggested to be involved in de novo biosynthesis while a basic isozyme, which predominates in muscle, appears to function in the purine nucleotide cycle. Antibodies specific for the basic isozyme detect a single protein in mouse tissues with highest levels in skeletal muscle, tongue, esophagus, and heart tissue consistent with a role for the enzyme in muscle metabolism. A series of degenerate oligonucleotides were constructed based on peptide sequences from purified rat muscle enzyme and then used to clone a mouse muscle cDNA encoding the basic isozyme. The clone contains a open reading frame of 1356 bases with 452 amino acids. Northern analysis of RNA from mouse tissues showed a tissue distribution similar to that of the protein, indicating a high level of gene expression in muscle. Transfection of COS cells with the mouse muscle cDNA allows expression of a functional protein with a molecular mass of approximately 50 kDa, consistent with the open reading frame and the size of the isolated rat enzyme. The deduced amino acid sequence of the mouse synthetase is 47 and 37% identical to the synthetase sequences from Dictyostelium discoideum and Escherichia coli, respectively. The availability of antibodies and cDNA clones specific for the basic isozyme of adenylosuccinate synthetase from muscle will facilitate future genetic and biochemical analysis of this protein and its role in muscle physiology.     

Molecular cloning and characterization of anther-preferential cDNA encoding a putative actin-depolymerizing factor

A cDNA clone, LMP131A, which is preferentially expressed in mature anther was isolated from a lily cDNA library. Northern blot analysis and plaque hybridization expriments showed that the LMP131A mRNA is present at ca. 0.3% of the mRNA in mature pollen and is not detectable in carpel, petal, floral bud, leaf, or root. The clone contains an open reading frame of 139 amino acid residues which shows greater than 40% sequence identity in a 91 amino acid overlap to animal actin-depolymerizing factors (ADF), cofilin and destrin. The sequences at and near the actin-binding site are most conserved. Using the lily clone as a probe, a cDNA clone, BMP1, was isolated from a mature anther library of Brassica napus. The expression pattern of the BMP1 clone was the same as that of the lily clone. The Brassica anther-preferential clone contains an open reading frame which is 79% identical to the lily LMP131A protein. Southern blot analysis showed that there are one or a few copies of the putative ADF genes in B. napus and Arabidopsis thaliana.     

Molecular cloning and characterization of a cDNA encoding soybean nodule IMP dehydrogenase

Inosine monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in de novo purine biosynthesis and is a postulated key enzyme in nitrogen assimilation in ureide-exporting nodules. A 2016 bp cDNA for IMPDH, designated as IMPDH, was cloned from a soybean nodule cDNA library. IMPDH encodes a polypeptide of 502 amino acids with a predicted molecular weight of 53000 and a pI of 5.54. The deduced IMPDH is 70.5% identical to that in Arabidopsis, with a 100% homology in the putative active-site region. Expressing the cloned cDNA in Escherichia coli mutant strain KLC381 (DeltaguaB) restored IMPDH activity, permitting bacterial growth on minimal medium. Southern blot analysis suggested a single copy of IMPDH gene in the soybean genome. Northern blot analysis showed that the expression of IMPDH gene is apparently nodule-specific.     

Molecular cloning and characterization of a tobacco leaf cDNA encoding a phosphate transporter

Phosphate acquired by roots is translocated to and utilized by the upper part of the plant, where the phosphate transport in the cell is also important in the phosphate metabolism. In order to study the role of the phosphate transporter in the regulation of the phosphate movement across the membranes in leaf cells, we isolated and characterized a 2,059 bp tobacco leaf cDNA clone, NtPT1. The 537 amino acid sequences, deduced from NtPT1, exhibited 93 and 91% identites to one of the high affinity phosphate transporters constitutively expressed in potato and tomato roots, respectively. The NtPT1 contains 12 membrane-spanning domain with a central hydrophilic region. The expression of NtPT1 in the yeast high affinity phosphate transporter mutant strain, NS219, complemented the mutant and promoted cell growth significantly. These results strongly suggest that NtPT1 encodes a functional phosphate transporter and that one of the high affinity phosphate transporters expressed in roots is also expressed in leaves. Southern analysis indicated that tobacco phosphate transporter genes are low copy number genes and members of a small multi-gene family.     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.