Cloning,expression and characterization of a novel human VMP gene*

We report here cloning and characterization of a novel human gene, termed VMP, which is a vesicular membrane protein. RT-PCR analysis shows that VMP is expressed exclusively in brain of the 16 tissues examined, suggesting that it is a neuron-specific membrane protein. The cDNA encodes 195 amino acid with a putative molecular weight of about 24 KDa. VMP contains two putative membrane spanning domains and a hydrophilic tail homologous to the microtubule-binding domain of MAPs. So it is speculated that VMP may associated with microtubules through its C-terminal and plays an important role in vesicular organelles transport and nerve signals.     

Cloning,expression, and initial characterization of a novel cytokine-like gene family

We report the identification and characterization of a novel cytokine-like gene family using structure-based methods to search for novel four-helix-bundle cytokines in genomics databases. There are four genes in this family, FAM3A, FAM3B, FAM3C, and FAM3D, each encoding a protein (224-235 amino acids) with a hydrophobic leader sequence. Northern analysis indicates that FAM3B is highly expressed in pancreas, FAM3D in placenta, and FAM3A and FAM3C in almost all tissues. Immunohistochemistry showed that FAM3A is expressed prominently in the vascular endothelium, particularly capillaries. We found that FAM3A and FAM3B protein were both localized to the islets of Langerhans of the endocrine pancreas. Recombinant FAM3B protein has delayed effects on beta-cell function, inhibiting basal insulin secretion from a beta-cell line in a dose-dependent manner.     

Cloning, expression and characterization of a novel human REPS1 gene.

Ral is a member of the small GTPase-binding protein (G protein) family, and plays an important role in the Ras-RalGDS signal transduction pathway. A series of recent findings reveal several important downstream target proteins of Ral, such as RalBP1, Reps1, and others. Here we report another binding partner for RalBP1, which we have isolated from the human fetal brain library. The human REPS1 protein shares 83% amino acid identity with the mouse Reps1 protein. Northern blot analysis shows that the REPS1 is expressed in a variety of tissues, with the strongest expression in the heart and testis.     

Cloning,expression and characterization of a novel aldehyde dehydrogenase gene from Flammeovirga pacifica

A novel putative aldehyde dehydrogenase (ALDH) gene aldh1413 from Flammeovirga pacifica isolated from deep sea sediment was cloned, expressed, and characterized. The molecular weight of the ALDH1413 (479 amino acids) was estimated by SDS-PAGE to be 53 kDa. The optimum temperature and pH for ALDH1413 were 35°C and 9.0, respectively. In the presence of either NAD⁺ or NADP⁺, the enzyme could oxidize a number of aliphatic aldehydes, particularly C3-and C5-aliphatic aldehydes and aromatic aldehydes such as benzaldehyde, which indicates that the enzyme belongs to broad-specific (ALDH) superfamily. Steady-state kinetic study revealed that ALDH1413 had a K _M value of 0.545 mM and a k _cat value of 7.48 s^?1 when propionaldehyde was used as the substrate. The Na⁺ could enhance ALDH1413 activity, which indicated it might be adapt to its habitat, marine environment.     

Cloning and expression of the large zebrafish protocadherin gene, Fat

The cadherin superfamily members play an important role in mediating cell-cell contact and adhesion (Takeichi, M., 1991. Cadherin cell adhesion receptors as a morphogenetic regulator. Science 251, 1451-1455). A distinct subfamily, neither belonging to the classical or protocadherins includes Fat, the largest member of the cadherin super-family. Fat was originally identified in Drosophila. Subsequently, orthologues of Fat have been described in man (Dunne, J., Hanby, A. M., Poulsom, R., Jones, T. A., Sheer, D., Chin, W. G., Da, S. M., Zhao, Q., Beverley, P. C., Owen, M. J., 1995. Molecular cloning and tissue expression of FAT, the human homologue of the Drosophila fat gene that is located on chromosome 4q34-q35 and encodes a putative adhesion molecule. Genomics 30, 207-223), rat (Ponassi, M., Jacques, T. S., Ciani, L., ffrench, C. C., 1999. Expression of the rat homologue of the Drosophila fat tumour suppressor gene. Mech. Dev. 80, 207-212) and mouse (Cox, B., Hadjantonakis, A. K., Collins, J. E., Magee, A. I., 2000. Cloning and expression throughout mouse development of mfat1, a homologue of the Drosophila tumour suppressor gene fat [In Process Citation]. Dev. Dyn. 217, 233-240). In Drosophila, Fat has been shown to play an important role in both planar cell polarity and cell boundary formation during development. In this study we describe the characterization of zebrafish Fat, the first non-mammalian, vertebrate Fat homologue to be identified. The Fat protein has 64% amino acid identity and 80% similarity to human FAT and an identical domain structure to other vertebrate Fat proteins. During embryogenesis fat mRNA is expressed in the developing brain, specialised epithelial surfaces the notochord, ears, eyes and digestive tract, a pattern similar but distinct to that found in mammals.     

Cloning, expression, purification, and characterization of zebrafish cytosolic serine hydroxymethyltransferase

A cDNA which encodes for zebrafish serine hydroxymethyltransferase (SHMT) has been cloned into a pET43.1a vector as a NdeI-EcoRI insert and transformed into HMS174(DE3) cells. After induction with isopropyl thiogalactoside, the enzyme was purified with a three-step purification protocol and about 15 mg of pure enzyme was obtained per liter of culture. Spectral and structural characteristics of the recombinant zebrafish SHMT are similar to the rabbit and human cytosolic SHMT. Kinetic constants for the natural substrates l-serine and tetrahydrofolate are also comparable to the values obtained previously for the rabbit and human cytosolic enzyme.     

Cloning, expression and characterization of gene sgcD involved in the biosynthesis of novel antitumor lidamycin

Lidamycin, an antitumor antibiotic composed of a macro-molecule peptide and enediyne chromophore[1] and originally named C1027, is produced by Streptomyces globisporus C1027 isolated from the soil in Qianjiang County, Hubei Province, China. It has extremely high antitu- mor activity, which has been proved to be the highest among antitumor compounds[2], being 1000- fold higher than that of adriamycin commonly used in clinic. The structure of lidamycin consists of an acid apoprotein and a chr…     

黑曲霉（Aspergillus niger）F044脂肪酶新型基因lipB的克隆、表达及酶学性质分析

摘要：【目的】本研究拟克隆新型的黑曲霉（Aspergillus niger）脂肪酶（EC 3.1.1.3）基因,实现其在大肠杆菌（Escherichia coli）的高效表达,并对表达产物进行系统的酶学性质分析,为该脂肪酶的工业化生产及应用奠定基础。【方法】通过PCR和RT-PCR克隆脂肪酶基因,并将其开放式阅读框（ORF）克隆入融合表达载体pET28a;表达产物经Ni-agarose纯化后对LipB进行酶学性质分析,并通过圆二色谱进行结构分析。【结果】成功地从A. niger F044中克隆了一个新型的脂肪酶基因lipB,获得了该基因的全基因组序列和cDNA序列（GenBank: FJ536287、FJ536288）,并实现了其在E. coli中的高效表达。LipB分子量约为43.0 kDa,最适底物为pNPC（C8）,酶学动力学常数Km=5.98 mmol/L,最适反应温度为50℃,最适pH为6.0;该酶能在40℃条件下保持稳定,在60℃条件下处理1 h后残余酶活仅为18.8%;该酶对Ca2+敏感,当脂肪酶经2 mmol/L Ca2+处理1 h后,酶活提高了2.6倍。圆二色谱分析表明该酶在Ca2+处理前后具有明显的结构变化。【结论】新型A. niger脂肪酶lipB基因的克隆不仅积累了脂肪酶基因资源,而且为高效基因工程菌的构建及规模化应用奠定基础;对LipB的酶学性质分析表明该酶在食品和油酯化工等领域具有广阔的应用前景。     

Cloning and developmental expression of a zebrafish meis2 homeobox gene

We show here that a zebrafish meis2 gene homolog has a dynamic expression pattern in the developing mesoderm and central nervous system. Meis family homeodomain proteins are known to act as cofactors with other homeodomain proteins. We find expression of meis2.1 in the developing zebrafish hindbrain and somites, correlating with reported sites of zebrafish hox gene expression, as well as in presumptive cerebellum, midbrain, retina and ventral forebrain. The expression pattern shares some, but not all, features with that of murine Meis2.     

Cloning and expression pattern of the lysozyme C gene in zebrafish

Here, we report isolation and developmental expression pattern of the zebrafish lysozyme C gene. Amino acid sequence analysis showed that the zebrafish lysozyme C protein shared approximately 37-80% identities with the mouse, human, chicken, and carp counterparts. Whole-mount in situ hybridization showed that the lysozyme C gene was expressed in macrophages, as its expression was co-localized with the known myeloid lineage markers L-plastin and PU.1. At 20 hours postfertilization (hpf), most of the lysozyme C positive cells were localized in the yolksac and head mesenchyme but not in the intermediate cell mass, supporting the notion that the primitive macrophage originated from the yolksac (Development 126 (1999) 3735). At 36hpf, the lysozyme C positive cells scattered within the head and yolksac, and began to appear in the caudal part of axial vein. By 6 days postfertilization (dpf), the lysozyme C positive cells accumulated in the kidney where hematopoiesis had been indicated to take place after 4dpf (Dev. Dyn. 214 (1999) 323). Taken together, our results demonstrate that the lysozyme C gene is specifically expressed in myeloid lineage, suggesting that it could serve as an excellent marker for genetic screening of both primitive and definitive myeloid lineage development in zebrafish.     

Molecular cloning,expression, and functional characterization of a novel zebrafish cytosolic sulfotransferase

By searching the zebrafish expressed sequence tag (EST) database, we have identified a cDNA clone encoding a putative zebrafish cytosolic sulfotransferase (ST). This cDNA was isolated and subjected to nucleotide sequencing. Analysis of the sequence data revealed that this novel zebrafish ST displays 32-35% amino acid sequence identity to members of all major cytosolic ST gene families. Therefore, this zebrafish ST, while belonging to the cytosolic ST gene superfamily, appears to be independent from all known constituent ST gene families. Recombinant zebrafish ST, expressed using the pET23c prokaryotic expression vector and purified from transformed Escherichia coli cells, migrated as a 34-kDa protein upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified zebrafish ST displayed sulfating activities toward dopamine and thyroid hormones (T(3) and T(4)), with a pH optimum spanning 7-9. The enzyme also exhibited activities toward a number of xenobiotics including some flavonoids, isoflavonoids, and other phenolic compounds. A thermostability experiment revealed the enzyme to be relatively stable over a temperature range between 20 and 48 degrees C. Among 10 divalent metal cations tested, Fe(++), Hg(++), Co(++), Zn(++), Cu(++), and Cd(++) exhibited dramatic inhibitory effects on the activity of the enzyme. These results constitute a first study on the cloning, expression, and characterization of a zebrafish cytosolic ST.     

The isolation, characterization, and expression of a novel GDF11 gene and a second myostatin form in zebrafish, Danio rerio

In the current study, the first non-mammalian growth/differentiation factor (GDF) 11-like homolog was cloned from zebrafish. At the nucleotide level, zebrafish GDF11 is most similar to human GDF11 (79%), while the peptide is most similar to mouse GDF11 (78%). Phylogenetic analysis showed that the zebrafish GDF11 clusters with mammalian GDF11s. This study also cloned a second MSTN form in zebrafish most similar to Salmonid MSTN2 forms. Based on real time PCR, GDF11 is expressed in multiple adult tissues, with levels highest in whole heads and gonads, and expression is less ubiquitous when compared to MSTN expression. During embryonic development, real time PCR demonstrated increasing GDF11 mRNA levels 10 h post-fertilization (hpf), while MSTN mRNA levels remain low until 48 hpf. This is the first report of a transforming growth factor (TGF)-beta superfamily member in a non-mammalian species that is more closely related to GDF11 than MSTN, and also a second form of MSTN in zebrafish; suggesting that a more complex TGF-beta superfamily array exists in primitive vertebrates than previously thought.     

Cloning,characterization, and heterologous expression of a novel glucosyltransferase gene from sophorolipid-producing Candida bombicola

Candida bombicola is well-studied for the production of a biosurfactant, the sophorolipids. In this paper, the cloning of a glucosyltransferase gene using polymerase-chain-reaction (PCR) technique is described. Degenerative primer-pairs were first designed based on the highly conserved amino-acid sequences of several selected yeast glucosyltransferases. Using these primers, an amplified sequence (amplicon) of 700 base-pair from C. bombicola was obtained and subsequently sequenced. Based on the sequence of this amplicon, additional target-specific PCR primers were designed for use in subsequent rounds of 3′- and 5′-extension using DNA walking technique to eventually obtain a C. bombicola genomic sequence containing an open-reading-frame putatively identified as a glucosyltransferase (gtf-1). The gene was subcloned in Saccharomyces cerevisiae for expression and functional characterization. Quantitative RT-PCR confirmed the expression of gtf-1 in the recombinant S. cerevisiae. In vitro assay with the sonicated cells of the recombinant yeast confirms the presence of glucosylation activity on sterol and hydroxy fatty acid substrates. This study reports for the first time the cloning and characterization of a broad-specificity lipid glucosylation gene from C. bombicola, and the functional activity of its gene product.