Molecular cloning and expression of a MAP kinase homologue from pea

The cdc2 kinases are important cell cycle regulators in all eukaryotes. MAP kinases, a closely related family of protein kinases, are involved in cell cycle regulation in yeasts and vertebrates, but previously have not been documented in plants. We used PCR to amplify Brassica napus DNA sequences using primers corresponding to amino sequences that are common to all known protein kinases. One sequence was highly similar to KSS1, a MAP kinase from Saccharomyces cerevisiae. This sequence was used to isolate a full-length MAP kinase-like clone from a pea cDNA library. The pea clone, called D5, shared approximately 50% amino acid identity with MAP kinases from yeasts and vertebrates and about 41% identity with plant cdc2 kinases. An expression protein encoded by D5 was recognized by an antiserum specific to human MAP kinases (ERKs). Messenger RNA corresponding to D5 was present at similar levels in all tissues examined, without regard to whether cell division or elongation were occurring in those tissues.     

Molecular cloning, characterization and expression analysis of an IL-21 homologue from Tetraodon nigroviridis

Interleukin-21 (IL-21) is an important immune cytokine that was well characterized in human and mammals, but little is known in fish. In present study, an IL-21 homologue was cloned and well characterized from Tetraodon nigroviridis. The full-length Tetraodon IL-21 cDNA was 849bp in size, containing an open reading frame (ORF) of 438bp that translated a 145 amino-acid peptide, a 5' untranslated region (UTR) of 69bp, and a 3' UTR of 342bp. The deduced peptide shared identity of 20-49% with other known IL-21 sequences. The Tetraodon IL-21 gene had six exons while both human and Takifugu IL-21 gene contained only five exons. However, the level of synteny between human, Takifugu and Tetraodon genomes was well conserved during evolution. In vivo expression study showed that Tetraodon IL-21 mRNAs were constitutively expressed at a low level and only in limited tissues, including gut, gill and gonad in healthy fish, and stimulation with LPS increased the expression of IL-21 in these tissues and induced the expression of IL-21 in kidney, spleen and skin, indicating that IL-21 is an inflammatory stress inducible gene associated with the anti-bacterial defense in fish. Our study provided further evidence for the existence of IL-21 in fish, and gained further insight into the immunological functions of IL-21 gene in fish.     

Molecular cloning and expression analysis of an HINT1 homologue from maize (Zea mays L.)

Histidine triad nucleotide binding protein (HINT1) belongs to a histidine triad (HIT) superfamily, which contains a highly conserved His-X-His-X-His-XX motif (X is a hydrophobic amino acid) and plays an important role in many biological processes. In this study, we have isolated the full-length cDNA of an HINT1 homologue from maize (Zea mays L.), designated as Zm-HINT1. The full-length cDNA of Zm-HINT1 consists of 729 bp with an ORF encoding a 138-amino acid protein. The deduced amino acid sequence of Zm-HINT1 shows high sequence homology to the mammalian HINT1 and contains conserved domains including the HIT motif, helical regions and β-strands, which are the characteristics of HINT1 proteins. The phylogenetic analysis has revealed that Zm-HINT1 is branched along with Caenorhabditis elegans HINT1. RT-PCR analysis has revealed that Zm-HINT1 is ubiquitously expressed in maize tissues but not in the pericarp, thus suggesting that Zm-HINT1 may not be related to the production of fibrin. Furthermore, expression levels of Zm-HINT1 have increased rapidly following treatment with salicylic acid. Taken together, these results indicate that Zm-HINT1 is a mammalian HINT1 homologue and may be involved in the immune response of maize.     

Molecular cloning,expression and antioxidant activity of a peroxiredoxin 2 homologue from Lampetra japonica

Peroxiredoxin (Prx) is a cellular antioxidant protein family that plays important roles in oxidative stress and immune cytotoxicity. In this study, we cloned a homologue of the Prx2 from the buccal gland of Lampetra japonica (L. japonica). L. japonica Prx2 (Lj-Prx2) contained two highly conserved motifs and shared more than 70% identity with the homologs from other vertebrate species. Phylogenetic analysis revealed that Lj-Prx2 is closely related to other available teleost Prx2. The real-time PCR results demonstrated that the Prx2 gene was widely expressed in adult lamprey. In addition, the expression of Prx2 gene was particularly up-regulated in red blood cells (RBCs) after the experimental animals were challenged with lipopolysaccharide (LPS) in vivo. Lj-Prx2 gene was subcloned into the pET23b vector and expressed in Escherichia coli BL21 (DE3). The recombinant L. japonica Prx2 (rLj-Prx2) was purified by using His Bind affinity chromatography. Polyclonal antibody to rLj-Prx2 was generated in New Zealand Rabbit. Western blot analysis showed that the Lj-Prx2 is present in the buccal gland secretion, suggesting the secretory feature of it. The function assays revealed that rLj-Prx2 has the capability to reduce the H₂O₂ when dithiothreitol (DTT) is used as a reducing equivalent and to protect DNA from oxidative damage. These findings suggested that Lj-Prx2 probably plays an essential role in antioxidant defense in RBCs to keep lamprey alive.     

Molecular cloning, characterization and expression of a novel trehalose-6-phosphate synthase homologue from Ginkgo biloba

In many organisms, trehalose acts as protective metabolite against harsh environmental stresses, such as freezing, drought, nutrient starvation, heat and salt. Herein a cDNA (designated as GbTPS, GenBank Accession Number AY884150) encoding a trehalose-6-phosphate synthase homologue was isolated and characterized from the living fossil plant, Ginkgo biloba, which is highly tolerant to drought and cold. GbTPS encoded an 868-amino-acid polypeptide with a predicted isoelectric point of 5.83 and molecular mass of 97.9 kD. Amino acid sequence alignment revealed that GbTPS shared high identity with class II trehalose-6-phosphate synthase homologues (67% identical to AtTPS7), but had only 17% and 23% of identity with OstA from Escherichia coli and ScTPS1 from S. cerevisiae, respectively. DNA gel blot analysis indicated that GbTPS belonged to a small multi-gene family. The expression analysis by RT-PCR showed that GbTPS expressed in a tissue-specific manner in G. biloba and might involve in leaf development. GbTPS was also found to be induced by a variety of stresses including cold, salt, drought and mannitol.     

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211bp, consisting of a 5'-terminal untranslated region (UTR) of 72bp, a 3'-terminal UTR of 77bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16h after injury and injection of bacteria.     

Calmodulin from Pharbitis nil: Purification and Characterization

A protein, identifiable as calmodulin (CaM), has been isolated from the seedling tissue of Pharbitis nil. The method has been developed to isolate a high quality protein from plant tissue containing the high content of polyphenols. This protein was relatively heat-stable and bound to hydrophobic resin in calcium-dependent manner. It was recognized by the antibody against pea and carrot, but did not bind to antibody against Dictyostelium discoideum. This protein had M_r of 15 kDa and 18.5 kDa in the presence and absence of Ca₂₊, respectively, and was able to stimulate calmodulin-deficient cAMP phosphodiesterase. Based on its migration on SDS-PAGE gels, M_r and binding to anti-CaM antibodies it was deduced that calmodulin from P. nil is essentially identical to calmodulin isolated from other plants.     

Molecular cloning of rat ATX1 homologue protein

An ATX1 homologue of 503 bp length was cloned from a rat cDNA library, and the deduced protein from the cDNA was found to contain 68 amino acids with a predicted molecular mass of 7.2 kDa. The rat ATX1 homologue protein (Rah1p), which shows 35%, 38%, and 89% identities with Atx1p, CUC-1, and HAH1, respectively, conserves both the MTCXXC copper-binding site in the N terminus and the KTGK lysine-rich region in the C terminus. In Northern blot analysis, rah1 mRNA was found to be expressed at high levels in the liver, small intestine, and testis. Expression of rah1 cDNA complemented a null atx1 mutant strain in yeast. Thus, Rah1p was concluded to be a functional copper chaperone.     

Molecular cloning and expression pattern of a Cubitus interruptus homologue from the mulberry silkworm Bombyx mori

Mutations in the human Crumbs homologue 1 (CRB1) gene cause severe retinal dystrophies. CRB1 is homologous to Drosophila Crumbs, a protein essential for establishing and maintaining epithelial polarity. We have isolated the mouse orthologue, Crb1, and analyzed its expression pattern in embryonic and post-natal stages. Crb1 is expressed exclusively in the eye, and the central nervous system. In the developing eye, expression of Crb1 is detected in the retinal progenitors, and later on becomes restricted to the differentiated photoreceptor cells where it remains active up to the adult stage. In the developing neural tube, expression of Crb1 is restricted to its most ventral structures, coinciding with the expression domain of Nkx2.2. In the adult brain, Crb1 expression is defined to areas where the production and migration of neurons occurs in adulthood.     

Cloning and structural analysis of the snap-back DNA of Pharbitis nil

We isolated and cloned DNA fragments that exist as inverted-repeat structures in the genome of Pharbitis nil. The method used exploited the fact that if inverted repeat DNA is present in the DNA fragment, intramolecular double-stranded structures can be partly formed within single-stranded DNA molecules after denaturation and rapid renaturation of the fragment. The rapidly renaturing DNA fragments (termed snap-back DNA) were isolated by hybroxylapatite column chromatography and treatment with mungbean nuclease and were cloned into the pUC9 vector. Four snap-back DNA members out of thousands of independent clones obtained were characterized with respect to the reiteration frequency and the nucleotide sequences. When used as probes in Southern hybridization experiments, some of the members identified restriction fragment length polymorphism among the cultivars, suggesting that these sequences might be fluid in the genome. One of the four clones has regions of nucleotide sequence homology to those of inverted-repeat regions in the transposon Taml of Antirrhinum majus.     

Molecular cloning and characterization of a human homologue of TBPIP, a BRCA1 locus-related gene

Here we clone the human homologue of TBPIP [Tat binding protein 1(TBP-1)-interacting protein]. TBPIP is a molecule that has been cloned from mouse as a cofactor of TBP-1. Eighty-eight per cent of the deduced amino acid sequence of human TBPIP coincides with that of mouse TBPIP. CAT assay reveals that human TBPIP could interact with human TBP-1, then enhance the function of TBP-1 on HIV (human immunodeficiency virus)-Tat-mediated transactivation. Our radiation hybrid mapping indicates that TBPIP is located on chromosome 17q12-21. A DNA database search uncovers that an apparent part of TBPIP has been obtained as a BRCA1 locus-related gene (OV-4) and mapped onto chromosome 17q12-21. Interestingly, the nucleotide structure of human TBPIP is very similar to that of the GT198 gene, which has been cloned from a human breast cancer cell line and also mapped onto the BRCA1 locus. Since a very high rate of gene mutation is observed in the BRCA1-related region in breast cancers and expression of authentic GT198 mRNA could not be confirmed in either BT-474 (other kind of human breast cancer cell line) or normal human testis (where the strong expression of GT198 mRNA is reported), it is likely that GT198 is a mutated form of human TBPIP.     

Neolignan and monoterpene glycoside from the seeds of Pharbitis nil

Phytochemical investigation of an EtOH extract of Pharbitis nil seeds (Convolvulaceae) resulted in the isolation and identification of a new neolignan, 7R,8S-threo-dihydroxydehydrodiconiferyl alcohol (1), and a new monoterpene glycoside, (3Z,7S)-7-hydroxy-3,7-dimethyl-3,8-octadienyl-β-d-glucopyranoside (2), together with a known compound, ethyl α-l-arabinofuranoside (3). The chemical structures of these compounds were unambiguously determined using physical data, HR-ESI–MS and spectroscopic evidence, including 1D and 2D NMR experiments. The anti-inflammatory activities of the isolates were evaluated by estimating the inhibition of nitric oxide (NO) production. Compounds 1 and 2 reduced NO levels in lipopolysaccharide (LPS)-stimulated murine microglial BV-2 cells. In addition, compound 2 showed weak cytotoxicity against the HCT-15 cell line with an IC₅₀ value of 28.6 μM.