Cloning and expression of a novel variant of human interferon-gamma cDNA

A cDNA library was prepared from the poly(A) mRNA isolated from human peripheral blood lymphocytes which were induced by combined treatment with phytohemagglutinin and a phorbol ester. Recombinant plasmids containing human interferon-gamma (HuIFN-gamma) cDNAs were identified by the oligonucleotide-hybridization method. Nucleotide sequence analysis showed that the nucleotide and amino-acid sequences of HuIFN-gamma cDNA in plasmid pIFN gamma-G4 differed from the published data at amino acid position 9 (CAA for glutamine versus AAA for lysine). The cDNA in plasmid pIFN gamma-G4 was expressed under control of the simian virus 40 early promoter in monkey COS cells and a biologically active HuIFN-gamma was secreted from the cells. The cDNA was also inserted into an expression vector carrying an E. coli tryptophan promoter and was expressed in E. coli. The results suggest that the conversion from lysine to glutamine at amino acid position 9 might not affect the specific activity of HuIFN-gamma.     

Cloning and characterization of osteoactivin, a novel cDNA expressed in osteoblasts.

Osteoblast development is a complex process involving the expression of specific growth factors and regulatory proteins that control cell proliferation, differentiation, and maturation. In this study, we used the rat mutation, osteopetrosis (op), to examine differences in skeletal gene expression between mutant op and normal littermates. Total RNA isolated from long bone and calvaria was used as a template for mRNA differential display. One of many cDNAs that were selectively expressed in either normal or mutant bone was cloned and sequenced and found to share some homology to the human nmb and Pmel 17 genes. This novel cDNA was named osteoactivin. Osteoactivin has an open reading frame of 1716 bp that encodes a protein of 572 amino acids with a predicted molecular weight of 63.8 kD. Protein sequence analysis revealed the presence of a signal peptide and a cleavage site at position 23. The protein also has thirteen predicted N-linked glycosylation sites and a potential RGD integrin recognition site at position 556. Northern blot analysis confirmed that osteoactivin was 3- to 4-fold overexpressed in op versus normal bone. RT-PCR analysis showed that osteoactivin is most highly expressed in bone compared with any of the other non-osseous tissues examined. In situ hybridization analysis of osteoactivin in normal bone revealed that it is primarily expressed in osteoblasts actively engaged in bone matrix production and mineralization. In primary rat osteoblast cultures, osteoactivin showed a temporal pattern of expression being expressed at highest levels during the later stages of matrix maturation and mineralization and correlated with the expression of alkaline phosphatase and osteocalcin. Our findings show that osteoactivin expression in bone is osteoblast-specific and suggest that it may play an important role in osteoblast differentiation and matrix mineralization. Furthermore, osteoactivin overexpression in op mutant bone may be secondary to the uncoupling of bone resorption and formation resulting in abnormalities in osteoblast gene expression and function.     

Cloning and expression of a cDNA encoding a novel human neurotrophic factor

A cDNA encoding a novel human neurotrophic factor (designated nerve growth factor-2; NGF-2) was cloned from a human glioma cDNA library using a synthetic DNA corresponding to human nerve growth factor (NGF). The cloned cDNA encodes a polypeptide composed of 257 amino acid residues including a prepro-sequence of 138 residues and a mature region of 119 residues. The amino acid sequence of human NGF-2 exhibits 58% similarity with that of human NGF. Conditioned medium of COS-7 cells transfected with an expression plasmid for human NGF-2 cDNA supported the survival of sensory neurons isolated from dorsal root ganglia of embryonic chicks. A 1.5 kb of NGF-2 mRNA can be detected from an early development stage in rat brain, by Northern blotting analysis.     

Cloning and characterization of a cDNA encoding a novel fibroblast growth factor preferentially expressed in human heart

A novel human fibroblast growth factor (hFGF), which shows 75% sequence homology with fibroblast growth factor-9, was isolated in random sequencing of a human heart cDNA library. The full-length sequence is 928 bp, the encoded protein is composed of 168 amino acid residues, and its pI value and molecular weight were estimated to be 8.13 and 19.1 kDa, respectively. RT-PCR using Marathon human heart cDNA shows that the coding region is approximately 507 bp. Southern hybridization showed a single band which indicates that this is a single copy gene. Northern hybridization done on a human multiple tissues blot showed that the gene is preferentially expressed in human heart, very weakly detectable in human brain and not detectable in 18 other different human tissues.     

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。     

ESX1L, a novel X chromosome-linked human homeobox gene expressed in the placenta and testis

A novel human homeobox gene related to the mouse Esx1 homeobox gene, which we have designated ESXR1, has been identified. ESXR1 and Esx1 share 65% identity within their homeodomains and have glutamic acid-rich and proline-rich N- and C-terminal regions, respectively. Unlike Esx1, ESXR1 contains 12 repeats of a unique nine amino acid motif, PPMAP(V/L)PPG, located C-terminal to the homeodomain. The general exon-intron structures of ESXR1 and Esx1 appear to be conserved. ESXR1 has been localized to human Xq22.1-q22.3, the same region of synteny shared by the map position of Esx1. ESXR1 expression appears to be restricted to the placenta and testis, the tissues in which Esx1 is also expressed. These data suggest that ESXR1 may be the orthologue of Esx1. The findings that there are similarities between ESXR1 and Esx1, yet differences between their encoded products, are consistent with the idea that placental genes evolve rapidly between mammalian species.     

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 expression of a human muscle phosphofructokinase cDNA

The nucleotide sequence of a 2.86-kb cDNA clone containing the complete human muscle phosphofructokinase (PFK) protein-coding region was determined. It comprises 76 bp of 5'-untranslated sequence, 2340 bp encoding human muscle PFK polypeptide, and 399 bp of 3'-untranslated sequence plus a poly(A) tract. A retroviral vector was utilized to express the product of this coding sequence in mouse fibroblasts. The PFK-coding cDNA was shown to code for an enzymatically active polypeptide by immunoprecipitation analysis and DEAE-Sephadex A-25 chromatography.     

Cloning and expression of a novel retinoblastoma binding protein cDNA,RBBP10

A 2860-bp cDNA was isolated from a human fetal brain cDNA library by high throughput cDNA sequencing, which encodes a putative protein with 186 amino acids. The putative protein shares 90.7% identity with rat pBOG (3403163) and shares 93.4% identity with human RBBP9 (NPA conserved RB binding domain, L × C × E, located between residue 63 and 68 was recognized. Therefore, it was named RBBP10. Mapviewer analysis locates it on human chromosome 20q11.22. RBBP10 spans about 9.6 kb of the genome and consists of six exons and five introns. RT-PCR revealed that the gene was expressed widely in various human tissues, and the expression level is somewhat higher in tumor tissues than in normal tissues. But subsequent sequencing analysis did not found any mutation of this in tumor tissues. The COS 7 cell transfected with the ORF of RBBP10 showed that the protein was distributed both in the cytoplasm and in the nucleus. Our results suggest that RBBP10 is the orthologue of the rat BOG gene (AF025819) and a paralogue of human RBBP9 (AF039564).     

T-SP1: a novel serine protease-like protein predominantly expressed in testis

Here, we describe a novel member in the group of membrane-anchored chymotrypsin (S1)-like serine proteases, namely testis serine protease 1 (T-SP1), as it is principally expressed in testis tissue. The human T-SP1 gene encompasses 28.7 kb on the short arm of chromosome 8 and consists of seven exons. Rapid amplification of cDNA ends (RACE) experiments revealed that due to alternative splicing three different variants (T-SP1/1, -2, -3) are detectable in testis tissue displaying pronounced heterogeneity at their 3'-end. T-SP1/1 consists of an 18 amino acid signal peptide and of a 49 amino acid propeptide. The following domain with the catalytic triad of His(108), Asp(156), and Ser(250) shares sequence identities of 42% and 40% with the blood coagulation factor XI and plasma kallikrein, respectively. Only T-SP1/1 contains a hydrophobic part at the C-terminus, which provides the basis for cell membrane anchoring. Using a newly generated polyclonal anti-T-SP1 antibody, expression of the T-SP1 protein was found in the Leydig and Sertoli cells of the testis and in the epithelial cells of the ductuli efferentes. Notably, T-SP1 protein was also detectable in prostate cancer and in some ovarian cancer tissues, indicating tumor-related synthesis of T-SP1 beyond testis tissue.     

Cloning, sequencing, and expression of cDNA for human beta-galactosidase

We cloned and sequenced the full-length cDNA for human placental beta-galactosidase. The 2379-nucleotide sequence contains 2031 nucleotides which encode a protein of 677 amino acids. The amino acid sequence includes a putative signal sequence of 23 amino acids and 7 potential asparagine-linked glycosylation sites. The cDNA in the expression vector pSVL was used to transfect COS cells. Expression of the cDNA in transfected COS cells produced immunoprecipitable proteins and led to an increase in beta-galactosidase activity.     

cDNA cloning and mRNA expression of a tandem-repeat galectin (PoGal2) from the pearl oyster, Pinctada fucata

Galectins can recognize and specifically bind to β-galactoside residues, playing crucial roles in innate immune responses of vertebrates and invertebrates. We cloned the cDNA of a tandem-repeat galectin from the pearl oyster Pinctada fucata (designated as PoGal2). PoGal2 cDNA is 1347 bp long and consists of a 5'-untranslated region (UTR) of 3 bp, a 3'-UTR of 297 bp with one cytokine RNA instability motif (ATTTA), and an open reading frame of 1047 bp, encoding a polypeptide of 349 amino acids, with an estimated molecular mass of 38.1 kDa and a theoretical isoelectric point of 8.5. PoGal2 contains two carbohydrate recognition domains (CRDs); both have the conserved carbohydrate-binding motifs H-NPR and WG-EE. PoGal2 shares 50.6 and 50.9% identity with those of abalone (Haliotis discus) and the Manila clam (Venerupis philippinarum), respectively. Phylogenetic analysis revealed that the tandem-repeat galectins formed two clades for the different species. Molluscan tandem-repeat galectins were clustered into a single clade, and nematode tandem-repeat galectins were clustered into another single clade. In both clades, CRD-N and CRD-C were divided into different groups. PoGal2 mRNA was constitutively expressed in all tissues analyzed, and the expression level of PoGal2 mRNA was found to be significantly up-regulated in digestive glands, gills and hemocytes after Vibrio alginolyticus stimulation/infection. Expression profile analysis showed that the expression level of PoGal2 mRNA was significantly up-regulated at 8, 12 and 24 h after V. alginolyticus infection. These results suggest that PoGal2 is a constitutive and inducible acute-phase protein involved in the innate immune response of pearl oysters.     

Cloning, characterization and genomic organization of LCC-1 (scya16), a novel human CC chemokine expressed in liver

By homology search of expressed sequence tags (EST) in GenBank a novel member of the CC chemokine family was identified. The full-length sequence of this liver-specific CC chemokine (LCC-1) predicted a mature protein of 97 amino acids with 31-48% identity to other CC chemokines. There was a characteristic amino acid C-term extension when aligned with other chemokines. Northern blot analysis from a panel of human tissues revealed that LCC-1 mRNA expression is restricted to adult and fetal liver. Different polyadenylation results in two mRNA species of 1.5 kb and 0.5 kb in size. LCC-1 is constitutively expressed in human HepG2 hepatoma cells and is induced by hypoxic exposure. The promoter region of the LCC-1 gene contains potential HIF-1 binding sites. The EST for LCC-1 has been previously mapped to the CC chemokine cluster on human chromosome 17q11.2. The organization of the LCC-1 gene (scya16) into three exons interrupted by two introns is identical to that found for other members of the CC chemokine family.     

Cloning and expression of a human ATP-citrate lyase cDNA.

A full-length cDNA clone of 4.3 kb encoding the human ATP-citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP-citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic-acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121,419 Da. Comparison of the human and rat ATP-citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP-binding site, as well as the CoA binding site. The human ATP-citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP-citrate lyase enzyme. Stable, amplified expression of ATP-citrate lyase in CHO cells as achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP-citrate lyase (3 pg/cell/d). Site-specific mutagenesis of His765----Ala diminishes the catalytic activity of the expressed ATP-citrate lyase protein. Since catalysis of ATP-citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP-citrate lyase.     

Cloning of human DECTIN-1, a novel C-type lectin-like receptor gene expressed on dendritic cells

We identified a new Ca2+-dependent lectin-like receptor gene, DECTIN-1 (HGMW-approved symbol CLECSF12), the human orthologue of mouse Dectin-1, coding for a putative type II transmembrane glycoprotein with an extracellular C-type lectin-like domain. The gene structure and two alternative spliced forms of DECTIN-1 are described. The DECTIN-1 gene was localized in the natural killer gene complex on human Chromosome 12p12.3-p13.2, between OLR1 and CD94 (position 21.8 cM on the genetic map). The DECTIN-1 gene is highly expressed at the mRNA level in dendritic cells and is not further up-regulated during the maturation of these cells with tumor necrosis factor-alpha. The DECTIN-1 gene therefore represents a novel human member of the C-type lectin-like receptor gene family preferentially expressed in dendritic cells.     

Cloning and functional expression of ATA1, a subtype of amino acid transporter A, from human placenta

This report describes the primary structure and functional characteristics of human ATA1, a subtype of the amino acid transport system A. The human ATA1 cDNA was isolated from a placental cDNA library. The cDNA codes for a protein of 487 amino acids with 11 putative transmembrane domains. The transporter mRNA ( approximately 9.0 kb) is expressed most prominently in the placenta and heart, but detectable level of expression is evident in other tissues including the brain. When expressed heterologously in mammalian cells, the cloned transporter mediates Na(+)-coupled transport of the system A-specific model substrate alpha-(methylamino)isobutyric acid. The transport process is saturable with a Michaelis-Menten constant of 0. 89 +/- 0.12 mM. The Na(+):amino acid stoichiometry is 1:1 as deduced from the Na(+)-activation kinetics. The transporter is specific for small short-chain neutral amino acids. The gene for the transporter is located on human chromosome 12.