Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis)

A tissue-specific cDNA library was constructed using polyA⁺ RNA from pituitary glands of the Indian catfishHeteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3′ region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5′ and 3′ untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence ofH. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.     

The b-32 protein from maize endosperm, an albumin regulated by the O2 locus: Nucleic acid (cDNA) and amino acid sequences

Summary The cDNA coding for the b-32 protein, an albumin expressed in maize endosperm cells under the control of the O2 and O6 loci, has been cloned and the complete amino acid sequence of the protein derived. A lambda gt11 cDNA library from mRNA of immature maize endosperm was screened for the expression of the b-32 protein using antibodies against the purified protein. One of the positive clones obtained was used to isolate a full-length cDNA clone. By Northern analysis, the size of the b-32 mRNA was estimated to be 1.2 kb. Hybrid-selected translation assays show that the message codes for a protein with an apparent molecular weight of 30–35 kDa. The nucleotide sequence shows that several internal repeats are present. The protein has a length of 303 amino acid residues (mol. wt. 32430 dalton) and its sequence shows the following features: no signal peptide is observable; it contains seven tryptophan residues, an amino acid absent in maize storage proteins; polar and hydrophobic residues are spread along the sequence; several pairs of basic residues are present in the N-terminal region; the secondary structure allows the prediction of two structural domains for the b-32 protein that would fold up giving rise to a globular shape. The cloning of this gene may help in understanding the role of the O2 and O6 loci in regulating the deposition of zein, the major storage protein of maize endosperm.     

Molecular Cloning of a cDNA Encoding Ribosome Inactivating Protein from Amaranthus viridis and Its Expression in E. coli

In order to isolate a cDNA clone of ribosome inactivating protein (RIP), a cDNA library was constructed in Uni-ZAP XL vector with poly(A) RNA purified from leaves of Amaranthus viridis. To get the probe for screening the library, PCR of phage DNA was conducted using the vector primer and degenerate primer designed from a conserved putative active site of the RIPs. Twenty-six cDNA clones from about 600,000 plaques were isolated, and one of these clones was fully sequenced. It was 1,047 bp and contained an open reading frame encoding 270 amino acids. The deduced amino acid sequence had a putative signal sequence of 17 amino acids and a putative active site (AIQMVAEAARFFKYIE) conserved in other RIPs. E. coli cells expressing A. viridis RIP cDNA did not grow well as compared to control cells, indicating that recombinant A. viridis RIP presumably inactivated E. coli ribosomes. In addition, recombinant A. viridis RIP cDNA produced by E. coli had translation inhibition activity in vitro.     

Characterisation of the gene encoding a protective antigen from Babesia microti identified it as η subunit of chaperonin containing T-complex protein 1

Passive immunisations with a monoclonal antibody termed 1-5H showed a partial but significant inhibition of parasitaemia against Babesia microti challenge infection. By immunoscreening with 1-5H, a clone (termed p58 gene) was obtained from a cDNA expression library of B. microti and the complete nucleotide sequence was determined. A protein homology search showed significant amino acid identities to the η subunit of the chaperonin containing T-complex protein 1 (CCT) of human (59%), mouse (58%) and Plasmodium falciparum (62%). Genomic analyses indicated that the p58 gene is present as a single copy gene and contains a total of approximately 400-bp introns in the genome of B. microti. The mAb 1-5H recognised a 58-kDa protein of B. microti and was found to cross-react with a 60-kDa protein of Babesia rodhaini. These results suggest the possibility that the p58 protein is the CCT η subunit of B. microti and functions as a chaperonin.     

Identification and Characterization of a cDNA Clone Encoding the Heat Shock Protein (Hsp60) from the Biting Midge, Culicoides variipennis sonorensis Wirth and Jones

A cDNA expression library constructed from Culicoides variipennis sonorensis was screened using an antibody specific for Hsp60 of Heliothis virescens. A single clone encoding the complete heat shock protein (Hsp60) of C. variipennis was identified and its 2400-bp insert was sequenced. The encoded 62-kDa protein contains 581 amino acids and includes a 26-amino acid putative mitochondrial targeting sequence at its N terminus and a GGM motif at its carboxyl terminus. Deduced amino acid sequences are highly similar (67–78%) to Hsp60 of other species, including the fruit fly, the house mouse, the Norwegian rat, the Chinese hamster, the human, a nematode, and the tobacco budworm moth. This is the initial isolation of a coding sequence for a stress-induced protein in C. variipennis.     

Cloning of a pea cDNA encoding a polypeptide of the light-harvesting complex associated with photosystem I using a monoclonal antibody

A monoclonal antibody (MAb UB42) is described that binds to thylakoids in pea chloroplasts, as shown by EM-immunogold labelling. The antibody recognised proteins of ca. 23–29 kDa in western blots of a pea leaf homogenate. A cDNA library was prepared from pea epidermal cells in the vector ZAP II, and immunoscreening of the library with UB42 led to the isolation of a clone, pUB42. This was sequenced and had an open reading frame of 269 codons encoding a predicted polypeptide of 28.9 kDa. The sequence showed extensive homology with three closely related polypeptides belonging to a family of chlorophyll a/b-binding proteins from the light harvesting complex of photosytem I (LHCI). Collectively, the results suggest that MAb UB42 recognises an epitope on the type II chlorophyll a/b-binding protein from LHCI and that clone pUB42 encodes this protein.     

Enzyme-coding genes as molecular clocks: The molecular evolution of animal alpha-amylases

Summary We constructed a cDNA library for the beetle,Tribolium castaneum. This library was screened using a cloned amylase gene fromDrosophila melanogaster as a molecular probe. Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids. Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results forD. melanogaster alpha-amylases, along with published sequences for other alpha-amylases. The results show that animal alpha-amylases are highly conserved over their entire length. A borader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals. We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.     

Isolation of a root-specific cDNA encoding a ns-LTP-like protein from the roots of bean (Phaseolus vulgaris L.) seedlings

A root-specific cDNA clone, PVR3, was isolated from a bean (Phaseolus vulgaris L.) root cDNA library by a differential screening procedure. The nucleotide sequence of PVR3 contains an open reading frame coding for an 11.14 kDa polypeptide of 102 amino acid residues; the first 25 amino acids correspond to the sequence characteristic of a signal peptide. Comparison of the deduced PVR3 polypeptide sequence with the polypeptide sequences of previously cloned genes indicates that PVR3 may encode a ns-LTP-like protein. Molecular modelling of the PVR3 protein predicts that it has a three-dimensional structure that is similar to the three-dimensional model determined from the maize ns-LTP. The PVR3 mRNA accumulated mainly in the roots of young seedlings. It can be detected at low levels in flowers, but it is not detected in other organs. Genomic Southern blot analysis indicates that the genomic DNA corresponding to PVR3 cDNA is encoded by a single gene or small gene family in the bean genome.     

Gene note. Cloning of a cDNA encoding the thaumatin-like protein of winter rye (Secale cereale L. Musketeer) and its functional characterization

A cDNA clone, WRTLP2, encoding an open reading frame of 173 amino acids, was recovered from a cDNA library of winter rye (Secale cereale L. Musketeer). The amino acid sequence deduced from the cloned cDNA exhibits very high sequence similarity (70-95%) with those of extracellular and low molecular weight thaumatin-like proteins of other cereals. It was possible to overexpress this isolated cDNA in Escherichia coli and it was found that the encoded protein of this clone exhibited antifungal activities against several fungal strains.     

Isolation,sequencing and analysis of the expression of Bryonia calmodulin after mechanical perturbation

A cDNA clone (Bc329) encoding calmodulin was isolated from a Bryonia cDNA library by screening with cloned Arabidopsis calmodulin cDNA. The cDNA Bc329 was 899 bp full-length clone. The predicted amino acid sequence consists of 149 residues and reveals a high homology with other known plant calmodulins (91 to 99% identity). Genomic southern blot suggests that Bryonia calmodulin is encoded by a single-copy gene. The Bc329 clone was used as a probe to study the expression of calmodulin mRNA after a mechanical stimulus applied on young Bryonia internodes. The steady-state of calmodulin mRNA reached a maximum 30 min after the treatment before it progressively decreased. The role of calcium and calmodulin as second messengers is discussed with regard to environmental changes.     

Molecular cloning,sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum

Protein sequence data derived from the N-terminal region of a 17 kDa polypeptide associated with the microsomal membrane fraction from Pisum sativum was used to design degenerate oligonucleotides which were used to amplify P. sativum cDNA via the polymerase chain reaction (PCR). Amplified cDNA was used as a probe to screen a P. sativum cDNA library and a cDNA clone, NDK-P1 was isolated and sequenced. The protein encoded by NDK-P1 had a calculated molecular mass of 16485 Da and possessed substantial homology with nucleoside diphosphate kinases (NDKs) isolated and cloned from other sources. High levels of expression of NDK-P1 protein were achieved in Escherichia coli using a T7-driven expression system. Recombinant NDK-P1 protein was shown to possess NDK activity and had similar biochemical characteristics to NDKs isolated from other sources. The Michaelis constants for a variety of nucleoside diphosphate (NDP) substrates were found to be broadly similar to those reported for other NDKs, with thymidine nucleotides being the sustrates of greatest affinity.     

Expression of a gene (Ids2) induced by Fe deficiency treatment in the roots of Hordeum vulgare L.

A ZAPII-cDNA library constructed from poly(A)⁺-RNA isolated from Fe-deficient barley roots was used for differential screening of barley roots grown in the presence and absence of Fe. Among seven clones that hybridised specifically to the probe for Fe deficiency, one clone (Ids2) was sequenced. Using a part of the cDNA sequence as a probe, a genomic-DNA library was probed and a corresponding DNA clone was isolated and sequenced. The predicted amino acid sequence resembled 2-oxoglutarate-dependent dioxygenase. Ids2 had a metal regulatory element as well as Cu regulatory elements of CUP1 gene of yeast MT.     

Isolation and characterization of a cDNA clone encoding the anti-viral protein from Phytolacca americana

Phytolacca anti-viral protein (PAP) was purified from Phytolacca leaves and the N-terminal was sequenced. A cDNA library was made from mRNAs isolated from Phytolacca leaves and cDNA clones for PAP were identified using oligonucleotide probes derived from the N-terminal amino acid sequence. The PAP-cDNA clone was sequenced from both directions. The predicted amino acid sequence of PAP was compared with the amino acid sequences of other ribosome-inactivating proteins. The identities of these proteins to PAP ranged from 29 to 38%, and a region was found in each with a sequence similar to the PAP sequence (AIQMVSEAARFKYI). Southern blot analysis indicates that PAP is encoded by a multi-gene family.Abbreviations MAP Mirabilis jalapa anti-viral protein - PAP Phytolacca anti-viral protein - SO6 30 kDa ribosome-inactivating protein from the seeds of Saponaria officinalis     

Purification and cloning of a novel antimicrobial peptide from salivary glands of the hard tick, Ixodes sinensis

A novel antimicrobial peptide named as ixosin-B was isolated from the salivary glands of the hard tick, Ixodes sinensis, by gel filtration, ion exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). Its amino acid sequence was determined as QLKVDLWGTRSGIQPEQHSSGKSDVRRWRSRY by Edman degradation. The cDNA encoding ixosin-B was cloned by cDNA library screening. The predicted protein from the cDNA sequence composed of 89 amino acids including mature ixosin-B. Purified ixosin-B exerted its antimicrobial activities against bacteria and fungi. No similarity was found by BLAST search to any database entries and, thus, our findings describe a novel antimicrobial peptide. It is also the fourth family of antimicrobial peptide from hard ticks.     

Molecular characterization of salt-stress-associated protein in citrus: protein and cDNA sequence homology to mammalian glutathione peroxidases

A gene encoding for a citrus salt-stress-associated protein (Cit-SAP) was cloned from Citrus sinensis salt-treated cell suspension. The gene, designated csa, was isolated from a cDNA expression library. The partial amino acid sequence of the protein, as well as that deduced from the nucleotide sequence of csa, revealed a considerable homology to mammalian glutathione peroxidase (GP), and to clone 6P229 from tobacco protoplasts. The increased expression of Cit-SAP in NaCl-treated cultured citrus cells and in citrus plants irrigated with saline water, and its similarity to GP, raise the possibility that one of the effects of salt stress in plants may be the increase of the level of free radicals.     

Isolation of a mRNA encoding a nucleoside diphosphate kinase from tomato that is up-regulated by wounding

A cDNA clone (TAB2) encoding a nucleoside diphosphate (NDP) kinase has been isolated from a tomato (Lycopersicon esculentum Mill. cv. Ailsa Craig) cDNA library. The clone is 590 bp long and exhibits a high degree of sequence identity with spinach NDP kinases I and II, Pisum sativum NDP kinase I, Arabidopsis thaliana NDP kinase, Drosophila melanogaster NDP kinase, Dictyostelium discoideum NDP kinase and human Nm 23-H1 and Nm23-H2. Northern analysis has revealed that the mRNA encoded by TAB2 is up-regulated in both leaf and stem tissue in response to wounding. The increase is apparent within 1 h of wounding and is not further elevated by application of ethylene. Southern blot analysis indicates that TAB2 is a member of a small gene family.     

Isolation of a cDNA encoding a homologue of actin from Porphyra yezoensis (Rhodophyta)

We report the nucleotide sequence of a cDNA encoding an actin from amarine red alga, Porphyra yezoensis Ueda. A cDNA clone wasisolated from a leafy gametophyte cDNA library and analyzed for the sequence.The clone contained an open reading frame for a protein of 373 amino acidswhichexhibits sequence similarity to known actins. The GC content of the thirdposition (83.9%) was much higher than that at the first (56.3%) and second(42.4%) positions. The actin forms a gene family in the P.yezoensis genome. Comparison of the deduced amino acid sequenceshowed higher similarity to the Florideophycidae Chondruscrispus (85%) than to the ProtoflorideophycidaeCyanidioschyzon merolae (70%). The mRNA was detected inboth the leafy gametophytes and filamentous sporophytes. The nucleotidesequence data reported in this paper will appear in theDDBJ/EMBL/GenBank databases under accession number AB039831.     

Cloning of a urochordate cDNA featuring mammalian short consensus repeats (SCR) of complement-control protein superfamily

Mammalian tumor necrosis factor (TNF)-α degenerate polymerase chain reaction (PCR) primers were used to amplify a probe from Botryllus schlosseri (colonial ascidian) allogeneic rejection-cDNA library. A PCR product (269 bp) was cloned and sequenced encoding an open reading frame (ORF) of 89 amino acids (aa). This clone, which revealed no similarity to TNF-α, but a substantial similarity to mammalian proteins featuring short consensus repeats (SCRs) of the complement control superfamily, was used to probe the rejection-cDNA library. Two partial cDNA clones were isolated and sequenced (Bs. 1, 846 bp; Bs.2, 712 bp). The longest ORF in clone Bs. 1 (which lacks the 5' end of the cDNA) predicts a protein of 251 aa, which differs from Bs.2 at six nucleotides and four aa. We compare the as similarity (up to 50.5%) of Bs.l with the SCR-region of mammalian complement factor H, apolipoprotein H, selectins, and complement receptors type 1 and type 2. A somatomedin B-like domain at the C-terminus of Bs. 1 deduced protein was also recorded. We propose that this mosaic and polymorphic botryllid sequence, featuring mammalian-like SCRs, might be an ancestral molecule in the evolution of the chordate's complement-control protein superfamily.     

Sequence of an oleocin cDNA from Brassica napus

Antibodies raised against purified rapeseed 19 kDa oleosin protein were used to screen an embryo-derived gt11 expression library from Brassica napus. A near full-length cDNA clone, BnV, was isolated. The 781 bp cDNA contained an open reading frame of 549 bp followed by an untranslated region of 222 pb and a poly(A) region of 10 bp. Comparisons between this cDNA and a different oleosin cDNA previously isolated from the same library showed high degrees of sequence similarity in the central domain region and in the 3 untranslated region. Sequence similarities between the derived protein sequence of this cDNA and all other known oleosin protein sequences are discussed.