Cloning and characterization of human CAGLP gene encoding a novel EF-hand protein.

The EF-hand proteins, containing conserved Ca2+ binding motifs, play important roles in many biological processes. Through data mining, a novel human gene, CAGLP (calglandulin-like protein) was predicted and subsequently isolated from human skeleton muscle. The open reading frame of CAGLP is 543 bp in length, coding a putative Ca2+ binding protein with four EF-hand motifs. The deduced amino acid sequence of CAGLP displays high similarity with Bothrops insularis snake protein calglandulin (80%). The results of PCR amplification using cDNA from 17 human tissues indicated that human CAGLP is expressed in prostate, thymus, heart, skeleton muscle, bone marrow and ovary. Functional CAGLP::EGFP (enhanced green fluorescent protein) fusion protein revealed that CAGLP accumulated through-out Hela cells. Western blot using anti-EGFP antibodies indicated that the CAGLP protein has a molecular weight of about 19 kD. A phylogenetic tree showed that CAGLP and calglandulin may be orthologous proteins representing a distinct group in the EF-hand proteins.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

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 and characterization of hGMEB1, a novel glucocorticoid modulatory element binding protein.

A 21-bp element called glucocorticoid modulatory element (GME) modulates the glucocorticoid receptor-mediated responses via the binding of an as yet poorly characterized transacting complex of proteins containing the 88-kDa GMEB1 and the 67-kDa GMEB2. Using heat shock protein 27 (HSP27) as bait in the yeast two-hybrid assay, we cloned a 1.83-kb cDNA encoding a novel 573-amino acid protein called human GMEB1 (hGMEB1). hGMEB1 possesses a KDWK domain, contains sequences almost identical (36/38) to three tryptic peptides of rat GMEB1 and shares 38% identity with rat GMEB2. hGMEB1 is ubiquitously expressed as a 85-kDa protein in all cell lines and tissues examined. In vitro translated hGMEB1 bound specifically to GME oligonucleotides yielding a complex of similar size to the complex obtained using rat liver nuclear extracts. Both complexes were supershifted with an antibody specific to hGMEB1. Co-immunoprecipitation experiments confirmed the in vivo interaction of HSP27 with hGMEB1.     

人类新基因cegl cDNA的克隆及表达研究

CLECT and EGF-like domain contained Gene 1(cegl)基因是用电子克隆的方法获得的人类新基因。该基因定位在人类的第14号染色体上,是一个单一外显子的基因。cegl基因的cDNA长度为2050bp,通过生物信息学方法预测它包含一个1340bp的完整阅读框架,编码一个490个氨基酸的蛋白,含有CLECT、EGF-like结构域各一个。以cegl基因全长编码区为探针的整体原位杂交结果显示该基因的小鼠和鸡的同源基因在各自早期胚胎头部中特异表达,并且在不同时期胚胎神经系统增殖迅速的部位中有大量的表达。RT-PCR结果显示该同源基因在小鼠成体各组织中广泛分布。这提示cegl基因可能与头部生长发育有密切关系,并且对维持成体各组织的正常功能起到重要的作用。对cegl基因在胚胎发育的时间和空间表达模式的研究将有助于进一步深入地揭示它在人脑的正常生长发育中的作用。     

Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein

We have cloned and characterized a new member of the phosphatidylinositol kinase (PIK)-related kinase family. This gene, which we term human SMG-1 (hSMG-1), is orthologous to Caenorhabditis elegans SMG-1, a protein that functions in nonsense-mediated mRNA decay (NMD). cDNA sequencing revealed that hSMG-1 encodes a protein of 3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by high nanomolar concentrations of wortmannin (IC(50) = 105 nm) but is not inhibited by a FKBP12-rapamycin complex. Mutation of conserved residues within the kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated in whole cells. Based on these data, we conclude that hSMG-1 is the human orthologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.     

Cloning and characterization of a novel human SPRYD4 gene encoding a putative SPRY domain-containing protein.

We report here the cloning and characterization of a novel human SPRYD4 gene which encodes a SPRY domain containing protein. The SPRYD4 gene is isolated from the human brain cDNA library, and mapped to 12q13.2 by searching the UCSC genomic database. The SPRYD4 cDNA is 1201 base pairs in length and contains an open reading frame encoding 207 amino acids. The SPRYD4 gene consists of two exons and encodes a putative protein with a SPRY domain ranging from 86 to 203 amino acids. The RT-PCR analysis reveals that SPRYD4 is ubiquitously expressed in 18 human tissues. However, it is strongly expressed in kidney, bladder, brain, thymus and stomach, while weakly expressed liver, testis, uterus, spleen and lung. Subcellular localization demonstrates that SPRYD4 protein is localized in the nuclear when overexpressed in COS-7 cell.     

The gene for histone RNA hairpin binding protein is located on human chromosome 4 and encodes a novel type of RNA binding protein.

The hairpin structure at the 3' end of animal histone mRNAs controls histone RNA 3' processing, nucleocytoplasmic transport, translation and stability of histone mRNA. Functionally overlapping, if not identical, proteins binding to the histone RNA hairpin have been identified in nuclear and polysomal extracts. Our own results indicated that these hairpin binding proteins (HBPs) bind their target RNA as monomers and that the resulting ribonucleoprotein complexes are extremely stable. These features prompted us to select for HBP-encoding human cDNAs by RNA-mediated three-hybrid selection in Saccharomyces cerevesiae. Whole cell extract from one selected clone contained a Gal4 fusion protein that interacted with histone hairpin RNA in a sequence- and structure-specific manner similar to a fraction enriched for bovine HBP, indicating that the cDNA encoded HBP. DNA sequence analysis revealed that the coding sequence did not contain any known RNA binding motifs. The HBP gene is composed of eight exons covering 19.5 kb on the short arm of chromosome 4. Translation of the HBP open reading frame in vitro produced a 43 kDa protein with RNA binding specificity identical to murine or bovine HBP. In addition, recombinant HBP expressed in S. cerevisiae was functional in histone pre-mRNA processing, confirming that we have indeed identified the human HBP gene.     

Cloning and characterization a novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase gene AGPAT7.

The 1-Acylglycerolphosphate acyltransferase is crucial enzyme for synthesis of glycerolipids as well as triacylglylcerol biosynthesis in eukaryotes. Six members of 1-acyl-sn-glycerol-3-phosphate acyltransferase family in human have been described, which were AGPAT1, 2, 3, 4, 5 and 6. Here we report the cloning and characterization of another novel human 1-acyl-sn-glycerol-3-phosphate acyltransferase member AGPAT7 (1-acyl-sn-glycerol-3-phosphate acyltransferase 7) gene, which was mapped to human chromosome 15q14. The AGPAT7 cDNA is 1898 bp in length, encoding a putative protein with 524 amino acid residues, which contains an acyltransferase domain in 123-234 aa. RT PCR amplification in 18 human tissues indicated that human AGPAT7 gene was widely expressed in uterus, thymus, pancreas, skeletal muscle, bladder, stomach, lung and testis. AGPAT7 protein was mainly localized to the endoplasmic reticulum (ER) in Hela cells.     

Cloning and characterization of a novel human TEKTIN1 gene

Tektins comprise a family of filament-forming proteins that are known to be coassembled with tubulins to form ciliary and flagellar microtubules. A new member of the tektin gene family was cloned from the human fetal brain cDNA library. We hence named it the human TEKTIN1 gene. TEKTIN1 cDNA consists of 1375 bp and has a putative open reading frame encoding 418 amino acids. The predicted protein is 48.3 kDa in size, and its amino acid sequence is 82% identical to that of the mouse, rat, and dog. One conserved peptide RPNVELCRD was observed at position number 323–331 of the amino acid sequence, which is a prominent feature of tektins and is likely to represent a functionally important protein domain. TEKTIN1 gene was mapped to the human chromosome 17 by BLAST search, and at least eight exons were found. Northern blot analysis indicated that TEKTIN1 was predominantly expressed in testis. By in-situ hybridization analysis, TEKTIN1 mRNA was localized to spermatocytes and round spermatids in the seminiferous tubules of the mouse testis, indicating that it may play a role in spermatogenesis.     

Crystallization and characterization of Pumilo: a novel RNA binding protein

Axis determination in early Drosophila embryos is controlled, in part, by regulation of translation of mRNAs transcribed in maternal cells during oogenesis. The Pumilio protein is essential in posterior determination, binding to hunchback mRNA in complex with Nanos to suppress hunchback translation. In order to understand the structural basis of RNA binding, Nanos recruitment, and translational control, we have crystallized a domain of the Drosophila Pumilio protein that binds RNA. The crystals belong to the space group P6(3) with unit cell dimensions of a = b = 94.5 A, c = 228.9 A, alpha = beta = 90 degrees, gamma = 120 degrees and diffract to 2.6 A with synchrotron radiation. We show that the purified protein actively binds RNA and is likely to have a novel RNA binding fold due to a very high content of alpha-helical secondary structure.     

Cloning and characterization of a novel human gene encoding a zinc finger protein with 25 fingers

This study reports cloning and characterization of a human cDNA encoding a novel human zinc finger protein, ZFD25. ZFD25 cDNA is 6118 bp long and has an open reading frame of 2352 bp that encodes a 783 amino acid protein with 25 C2H2-type zinc fingers. The ZFD25 cDNA also contains a region with high sequence similarity to the Krüppel-associated box A and B domain in the 5'-untranslated region, suggesting that ZFD25 belongs to the Krüppel-associated box zinc finger protein family. The ZFD25 gene was localized to chromosome 7q11.2. Northern blot analysis showed that ZFD25 was expressed in a wide range of human organs. In cultured endothelial cells, the mRNA level was decreased upon serum starvation.     

Identification of a novel HIV-1 TAR RNA bulge binding protein.

The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

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.