Molecular cloning and RNA expression of a novel Drosophila calpain,Calpain C

The calpains are Ca(2+)-activated cysteine proteases whose biochemical properties have been extensively characterized in vitro. Less is known, however, about the physiological role of calpains. In this respect, Drosophila melanogaster is a useful experimental organism to study calpain activity and regulation in vivo. The sequencing of the fly genome has been recently completed and a novel calpain homologue has been identified in the CG3692 gene product. We embarked on the cloning and characterization of this putative novel calpain. We demonstrate that the actual calpain is different from the predicted protein and we provide experimental evidence for the correction of the genomic annotation. This novel protein, Calpain C, must be catalytically inactive, having mutated active site residues but is otherwise structurally similar to the other known fly calpains. Moreover, we analysed Calpain C RNA expression during Drosophila development by RT-PCR and RNA in situ hybridization, which revealed strong expression in the salivary glands.     

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 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,biochemical and structural analysis of elongation factor-1 alpha from Leishmania donovani: comparison with the mammalian homologue

The Src-homology 2 domain containing protein tyrosine phosphatase-1 (SHP-1) is involved in the pathogenesis of infection with Leishmania. Recently, we identified elongation factor-1 alpha (EF-1 alpha) from Leishmania donovani as a SHP-1 binding and activating protein [J. Biol. Chem. 277 (2002) 50190]. To characterize this apparent Leishmania virulence factor further, the cDNA encoding L. donovani EF-1 alpha was cloned and sequenced. Whereas nearly complete sequence conservation was observed amongst EF-1 alpha proteins from trypanosomatids, the deduced amino acid sequence of EF-1 alpha of L. donovani when compared to mammalian EF-1 alpha sequences showed a number of significant changes. Protein structure modeling-based upon the known crystal structure of EF-1 alpha for Saccharomyces cerevisiae-identified a hairpin loop present in mammalian EF-1 alpha and absent from the Leishmania protein which corresponded to a 12 amino acid deletion. Consistent with these structural differences, the sub-cellular distributions of L. donovani EF-1 alpha and host EF-1 alpha were strikingly different. Interestingly, infection of macrophages with L. donovani caused redistribution of host as well as pathogen EF-1 alpha. Since EF-1 alpha is essential for survival, the distinct biochemical and structural properties of Leishmania EF-1 alpha may provide a novel target for drug development.     

Molecular cloning, overexpression, and purification of a major xylanase from Aspergillus oryzae

The gene encoding xylanase G2 (xynG2) was isolated from a genomic library of Aspergillus oryzae KBN616, used for making shoyu koji. The structural part of xynG2 was found to be 767 bp. The nucleotide sequence of cDNA amplified by RT-PCR showed that the open reading frame of xynG2 was interrupted by a single intron which was 71 bp in size and encoded 232 amino acids. Direct N-terminal amino acid sequencing showed that the precursor of XynG2 had a signal peptide of 44 amino acids. The predicted amino acid sequence of XynG2 has strong similarity to other family 11 xylanases from fungi. The xynG2 gene was successfully overexpressed in A. oryzae and the overpexpressed XynG2 was purified. The molecular weight of XynG2 estimated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 21,000. This was almost the same as the molecular weight of 20,047 calculated from the deduced amino acid sequence. The purified XynG2 showed an optimum activity at pH 6.0 and 58 degrees C. It had a Km of 5.1 mg/ml and a Vmax of 123 micromol/min/mg when birch wood xylan was used as a substrate.     

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.     

Molecular cloning and expression analysis of a CONSTANS homologue,PnCOL1, from Pharbitis nil

The Arabidopsis CONSTANS (CO) gene is a key regulator of the long day (LD)-dependent flowering pathway and two CO homologous genes COL1 and COL2 are involved in the regulation of the circadian rhythm. In order to understand the role of CO and COL in short-day plants, a CO homologue, PnCOL1, was isolated and characterized from Japanese morning glory (Pharbitis nil). The deduced PnCOL1 protein of 386 amino acid residues contained two putative zinc finger motifs at the N-terminal region and a conserved CCT domain at the C-terminal region. The deduced amino acid sequence of PnCOL1 was 34% identical to that of PnCO, but 32%, 34%, and 34% identical to those of CO, COL1, and COL2, respectively. Expression of PnCOL1 was barely detected in the cotyledons of plants grown under continuous light (CL), but highly expressed in the cotyledons of plants grown under SD. Expression of PnCOL1 showed a pattern of circadian rhythm as well as daily oscillation. The overexpression of PnCOL1 by a 35S promoter did not overcome the late-flowering phenotype of Arabidopsis co mutants. The results provided in this study suggest that PnCOL1 may have a role in the circadian rhythm in Pharbitis nil.     

Molecular cloning and expression of a mammalian homologue of a translationally controlled tumor protein (TCTP) gene from Penaeus monodon shrimp

We previously observed secretion of native-type Streptomyces mobaraensis transglutaminase (MTGase) in Corynebacterium glutamicum by co-expressing the subtilisin-like protease SAM-P45 from S. albogriseolus which processes the pro-region. In the present study, we have used a chimeric pro-region consisting of S. mobaraensis and Streptomyces cinnamoneus transglutaminases for the production of MTGase in C. glutamicum. As a result, secretion of MTGase using the chimeric pro-region is increased compared to that using the native pro-region.     

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, expression, and uses of mammalian glycosyltransferases.

Lineage specific expression patterns of mammalian oligosaccharides are regulated largely by glycosyltransferases. Several distinct glycosyltransferase genes have recently been isolated by gene transfer and traditional cloning approaches. These each maintain a type II transmembrane topology, that yields a COOH-terminal, Golgi-resident catalytic domain. However, significant primary sequence similarities between cloned glycosyltransferases have been found only between enzymes with substantial functional similarity. These cloned DNA segments, and their cognate enzymes, have been used to determine glycosyltransferase expression patterns, characterize the molecular basis for mutant glycosyltransferase alleles, and identify oligosaccharide ligands for members of the Selectin family of cell adhesion molecules. They have also shown promise for use in the preparative synthesis of defined oligosaccharide molecules, and for transgenic animal approaches designed to explore the function(s) of oligosaccharides in mammalian organisms.     

Molecular cloning of the dog homologue of the lymphocyte antigen CD28

The nucleotide data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number L22178.     

Molecular cloning of the human homologue to the pig protein-tyrosine kinase syk

The nucleotide sequence data reported in this paper have been submitted to the EMBL nucleotide sequence database and have been assigned the accession number X73568.     

鲈鱼钙蛋白酶基因cDNA克隆及表达分析

钙蛋白酶超家族（Calpain,CAPN,EC3.4.22.17）是由一类钙依赖性半胱氨酸蛋白酶组成.其在细胞周期、细胞迁移、信号转导以及细胞凋亡中都有着广泛的作用.研究了鲈鱼催化亚基CAPN2的基因结构.鲈鱼（Lateolabrax japonicus） CAPN2 cDNA全长2373 bp,5′非翻译区74 bp、3′非翻译区205 bp、开放阅读框2094 bp,编码一个由697个氨基酸组成的蛋白质,分子量为77.94kDa,等电点为5.02.氨基酸序列分析表明,鲈鱼CAPN2具有较高的保守性,与大西洋鲑（Hippoglossus hippoglossus）、虹鳟（Oncorhynchus mykiss）、斑马鱼（Danio rerio）、斑点叉尾鮰（Ictalurus punctatus）、猪（Sus scrofa）、人（Homo sapiens）、大鼠（Mus musculus）等 7个物种的同源性分别为89%、81%、77%、75%、63%、62%、62%.用RT-PCR分析CAPN2基因在10个组织中均有表达,肌、心、肾表达较高,脑、鳃、肠次之,肝、眼、脂肪、脾表达最低.     

Molecular cloning and characterization of a novel rat CXC chemokine, rPBP, the homologue of human and mouse PBP

We have previously cloned the mouse platelet basic protein (mPBP), a homologue of human PBP, from mouse thymic stromal cells. Using EST alignment and RT-PCR, the rat homologue of human and mouse PBP was cloned from lung and named as rPBP. The complete open reading frame and part of the 3'- and 5'-non-coding regions were obtained through rapid amplification of cDNA ends. The rPBP cDNA encodes a protein of 111 amino acids containing a signal peptide of 37 amino acids at the N-terminus, with the mature protein of 74 amino acids. The rPBP is a new member of ELR+CXC chemokines. The mature protein of rPBP shares 69% and 45% homology with mouse and human PBP, respectively. In situ hybridization assay revealed rPBP to be predominantly localized in the pulmonary vascular endothelial cells. The eukaryotic expression vector pCDNA3-rPBP was constructed and transiently transfected into COS-7 cells. In the in vitro chemotaxis assay, the polymorphonuclear leukocytes (PMNs) were chemoattracted to the supernatants from transfected COS-7 cells in a dose-dependent manner. The implication of rPBP found in rat lung is that this chemokine may have the function to recruit PMNs to fight against pulmonary infection.     

Molecular cloning of a cDNA fromBrassica napus L. for a homologue of acyl-CoA-binding protein

A cDNA encoding an acyl-CoA-binding protein (ACBP) homologue has been cloned from a gt11 library made from mRNA isolated from developing seeds of oilseed rape (Brassica napus L.). The derived amino acid sequence reveals a protein 92 amino acids in length which is highly conserved when compared with ACBP sequences from yeast, cow, man and fruit fly. Southern blot analysis ofBrassica napus genomic DNA revealed the presence of 6 genes, 3 derived from theBrassica rapa parent and 3 fromBrassica oleracea. Northern blot analysis showed that ACBP genes are expressed strongly in developing embryo, flowers and cotyledons of seedlings and to a lesser extent in leaves and roots.