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At least 685 records · Page 38Linked to original sources
PubMed1989

Nucleotide sequence and expression of the small (S) RNA segment of Maguari bunyavirus.

The small (S) RNA segment of the Maguari bunyavirus genome has been cloned as cDNA and its nucleotide sequence determined. The nucleocapsid protein, N, (Mr 26K) and a nonstructural protein, NSs, (Mr 11K), are encoded in overlapping reading frames, similar to other bunyavirus S RNA segments. In addition, a third AUG-initiated open reading frame encoding a 9.3K protein was observed. All three polypeptides were translated in cell free systems programmed with RNA transcribed in vitro from the cDNA subcloned downstream of a bacteriophage T7 promoter. The effects on expression of subcloning parts of the cDNA and by site-specific mutagenesis are discussed in relation to the scanning model of initiation of translation. A recombinant baculovirus has been constructed to express the Maguari virus S segment gene products. The N protein was efficiently expressed in infected cells, and a significant amount was in a soluble form. We could not detect the synthesis of NSs nor the 9.3K protein, and the reasons for this are discussed. The 9.3K protein has not been found in Maguari virus-infected cells and so the question of its functional significance remains open.

Amino Acid Sequence↗
PubMed1996

Expression of the major capsid protein of human papillomavirus type 11 in Saccharomyces cerevisae.

The major capsid protein L1 of papillomaviruses expressed recombinantly or in infected cells has the intrinsic ability to form virus-like particles (VLPs) which display conformational epitopes necessary to elicit high-titered, virus-neutralizing antibodies. We have shown previously that the L1 gene of human papillomavirus type 6a (HPV6) can be expressed efficiently in Saccharomyces cerevisae (Sc) as VLPs. However, when we attempted to express the L1 gene cloned from the closely related HPV11 in yeast, few VLPs were observed in crude lysates. The lower expression level of HPV11 L1 protein was found to result from a truncation of the HPV11 L1 mRNA. Since sequence requirements for transcriptional termination in yeast are still unclear, the HPV6 L1 gene was used as the basis for the complete synthetic reconstruction of the entire 1506-bp HPV11 L1 gene. Expression of this HPV6/11 hybrid L1 gene in yeast resulted in predominantly full-length L1 mRNA and a > 7-fold increased level of production of HPV11 VLPs compared to that expressed by the wild-type HPV11 L1 gene. The VLPs were shown to display the conformational epitopes important to elicit virus-neutralizing antibodies.

Base Sequence↗
PubMed1997

Transgene rescue in the mammary gland is associated with transcription but does not require translation of BLG transgenes.

Many transgenes, particularly those comprising cDNA sequences fail to be expressed when they are introduced into transgenic mice. We have previously shown that this problem can be overcome in the mammary gland by co-integrating a poorly expressed cDNA transgene, comprising the sheep beta-lactoglobulin promoter, with the efficiently expressed, unmodified beta-lactoglobulin gene. In this report we demonstrate that the transcription of the beta-lactoglobulin gene is associated with this effect because co-integration with a non-transcribed beta-lactoglobulin gene fails to rescue expression. By contrast, co-integration with a translationally inactivated beta-lactoglobulin transgene does rescue the expression of the second gene, but without the co-production of beta-lactoglobulin protein.

Animals↗
PubMed2003

Expression and characterization of Asp fI, an immunodominant allergen/antigen of A. fumigatus in insect cell.

Asp fI is a major allergen/antigen/cytotoxin of Aspergillus fumigatus and exhibits ribonuclease activity. This allergen plays a role in allergic and invasive Aspergillosis and reported as a major cytotoxin with ribonuclease activity. To express the protein in large quantity and to characterize the multifunctional nature of Asp fI, we have generated recombinant baculovirus by introducing the gene in pFastBac HTa expression vector and expressed in insect cell. The baculovirus expression vector system has been used as a versatile system for the efficient expression of proteins with most eukaryotic posttranslational modification. Recombinant Asp fI was expressed as approximately 1% of the total cellular protein in infected Sf9 insect cells. The protein was purified using Ni2+ affinity column chromatography and the yield of purified protein was approximately 10 mg/l g of total cellular protein. Immunoreactivity of the protein was determined by immunoblot analysis using both poly His monoclonal antibody, IgG and IgE antibodies present in the sera of ABPA patients. The protein was glycosylated as revealed by the glycoprotein staining and was observed to retain both ribonuclease and cytotoxic activities. These results suggest that Asp fI expressed in insect cell was post translationally modified and biologically active that can be used as a diagnostic marker for biochemical studies.

Allergens↗
PubMedDate not supplied

Requirement for an upstream element for optimal transcription of a bacterial tRNA gene.

Bacterial promoters are the sites at the 5' end of each gene that bind RNA polymerase and direct the initiation of transcription. The functional elements of Escherichia coli promoters are two highly conserved sequences, each about six nucleotides long, usually centred at sites -10 and -35, +1 being the initiating nucleotide. We have been interested in the structure of promoters of genes that are subject to stringent control, that is whose expression is reduced in conditions of amino acid shortage, such as rRNA and tRNA genes. We have therefore mapped the sequences involved in promoting in vivo transcription of a bacterial tRNATyr (tyrT) gene by fusing the tyrT promoter region to a galactokinase (galK) gene, and using in vivo expression of galactokinase activity to measure promoter strength. We show here that efficient expression from the tyrT promoter requires specific sequences upstream of the canonical promoter elements, and we suggest that these sequences constitute an extended promoter structure.

Base Sequence↗
PubMed1997

Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector.

The development of methods for efficient gene transfer to terminally differentiated retinal cells is important to study the function of the retina as well as for gene therapy of retinal diseases. We have developed a lentiviral vector system based on the HIV that can transduce terminally differentiated neurons of the brain in vivo. In this study, we have evaluated the ability of HIV vectors to transfer genes into retinal cells. An HIV vector containing a gene encoding the green fluorescent protein (GFP) was injected into the subretinal space of rat eyes. The GFP gene under the control of the cytomegalovirus promoter was efficiently expressed in both photoreceptor cells and retinal pigment epithelium. However, the use of the rhodopsin promoter resulted in expression predominantly in photoreceptor cells. Most successfully transduced eyes showed that photoreceptor cells in >80% of the area of whole retina expressed the GFP. The GFP expression persisted for at least 12 weeks with no apparent decrease. The efficient gene transfer into photoreceptor cells by HIV vectors will be useful for gene therapy of retinal diseases such as retinitis pigmentosa.

Animals↗
PubMed1986

Cell surface expression of class II histocompatibility antigens occurs in the absence of the invariant chain.

The invariant chain is a glycoprotein transiently associated with the alpha and beta subunits of class II antigens of the major histocompatibility complex during their transport to the cell surface. An expression assay with cDNA clones transfected into simian COS cells was used to test whether the invariant chain is required for assembly and transport of human class II antigens. COS cells do not express detectable levels of RNA from the endogenous invariant chain gene. Cell surface expression of the DP, DQ, and DR antigens was observed in COS cells transfected with the respective alpha and beta chain cDNA clones. Analysis of RNA from the transfected cells showed that the human genes were transcribed in COS cells and that the endogenous simian class II and invariant chain genes were not induced. Cotransfections with an invariant chain cDNA clone did not alter the levels of class II antigens at the cell surface. Biosynthetic labeling and immunoprecipitation demonstrated that the invariant chain cDNA was expressed into a protein which associated with DR alpha and beta chains. Efficient expression of DR antigen in absence of invariant chain was also observed at the surface of a human fibroblast line stably transfected with DR alpha and beta cDNA. This study demonstrates that expression of all three human class II antigens can be achieved with cDNAs cloned in expression vectors. Furthermore, cell surface expression of class II major histocompatibility complex antigens can occur in absence of invariant chain. The postulated role of the invariant chain in class II antigen transport to the cell surface must be reevaluated. The invariant chain may rather be involved in functional properties of class II molecules such as antigen presentation.

Antigens, Differentiation, B-Lymphocyte↗
PubMed1997

Human endothelin receptors ET(A) and ET(B) expressed in baculovirus-infected insect cells--direct application for signal transduction analysis.

We expressed human endothelin receptors, ET(A) and ET(B), in insect Sf9 cells infected by recombinant baculoviruses that contained the respective cDNAs. Ligand-binding experiments showed that the two expressed receptors have the same affinities as observed for the receptors in mammalian cells, i.e. the ET(A) receptor showed an affinity order of ET-1 > or = ET-2 >> ET-3, and the ET(B) receptor remained nonselective for three isopeptide ligands. The ET(B) receptor was purified by affinity chromatography with K9-biotinyl-ET-1 without losing the ligand-binding activity from the membrane of infected Sf9 cells. Protein chemical analysis of the purified ET(B) receptor showed that it is glycosylated, and that the N-terminal 38-amino-acid peptide is susceptible to proteolytic digestion, resulting in a small 35-kDa receptor like that found in the human placenta. Surprisingly, the infected and unlysed cells showed a strong intracellular Ca2+ concentration increase ([Ca2+]i), which was generated by a unique signal-transduction pathway consisting of the insect GTP-binding protein and human endothelin receptors expressed in the late phase of virus infection. Due mainly to an efficient expression (over 200,000 receptors/cell), to a low background owing to no endogenous homolog receptor in insect Sf9 cells, and to a sensitive fluorescent reagent Fura-2, this insect Sf9 cell system can detect the [Ca2+]i induced by picomolar levels of endothelin-receptor. We propose that this highly sensitive system be used to screen for potential antagonists/agonists of endothelin receptors.

Amino Acid Sequence↗
PubMed1988

The promoter for a sporulation gene in the spoIVC locus of Bacillus subtilis and its use in studies of temporal and spatial control of gene expression.

We have identified the transcription start site and regulatory region governing the expression of a sporulation gene in the spoIVC locus of Bacillus subtilis. Efficient expression and developmental regulation of this gene was controlled from a promoter region that extended no more than 110 base pairs upstream and no more than 4 base pairs downstream from the start site of transcription, on which basis we infer that spoIVC is regulated at the level of transcription initiation. Using a transcriptional fusion of the spoIVC gene to the lacZ gene of Escherichia coli, we found that spoIVC expression was turned on at the third to fourth hour of sporulation (at about the developmental stage [IV] that its products are required in spore formation) and that this transcription was largely restricted to the mother cell chamber of the sporangium. Mutations in many different spo genes (causing blocks at stages 0 to V) were found to influence (negatively and positively) the level of spoIVC expression. Our results distinguish the mode of spoIVC regulation from that of previously studied sporulation genes and indicate that it is representative of a new regulon of mother cell-specific gene expression.

Bacillus subtilis↗
PubMed1989

Cloning, characterization, and heterologous expression of the Saccharopolyspora erythraea (Streptomyces erythraeus) gene encoding an EF-hand calcium-binding protein.

The regulatory effects of Ca2+ in eucaryotic cells are mostly mediated by a superfamily of Ca2+-binding proteins (CABs) that contain one or more characteristic Ca2+-binding structural motifs, referred to as EF hands. We have cloned and sequenced the structural gene for an authentic EF-hand CAB from the spore-forming gram-positive bacterium Saccharopolyspora erythraea (formerly Streptomyces erythraeus). When the gene was introduced into Streptomyces lividans on the high-copy plasmid vector pIJ702, CAB was found to be expressed at higher levels than in S. erythraea, with no apparent effects on either growth or sporulation. A more convenient expression system for CAB was obtained by introducing an NdeI site at the initiation codon by using oligonucleotide-directed mutagenesis and placing the gene in the expression vector pT7-7 in Escherichia coli. In this system, CAB was efficiently expressed at levels up to 20 to 30% of total cell protein. When purified to homogeneity from either E. coli or Streptomyces lividans, CAB was found to be identical to the protein previously obtained from S. erythraea.

Amino Acid Sequence↗
PubMed2000

Constitutively active mitogen-activated protein kinase kinase increases GLUT1 expression and recruits both GLUT1 and GLUT4 at the cell surface in 3T3-L1 adipocytes.

To address a role of mitogen-activated protein kinase (MAPK) in the regulation of glucose transport, we made a constitutively active mutant of MAPK kinase (MAPKK) and introduced it into 3T3-L1 preadipocytes by using a retrovirus-mediated transfection procedure. The deletion of 20 amino acids (those between and including 32 and 51) in the amino terminal region of Xenopus MAPKK and the replacement of serine residues on the 218 and 222 positions by glutamic acid (dSESE-MAPKK) let Xenopus MAPKK constitutively active. The isolated cell clones differently expressing dSESE-MAPKK (clone 219 higher expression, clone 233 lower expression) efficiently differentiated to adipocytes by a standard differentiation cocktail. Accordingly, the increased expression of dSESE-MAPKK protein during differentiation resulted in the increased basal MAPK activity in clone 219 adipocytes and, to a lesser extent, in clone 233 adipocytes. In contrast to clone 233 and parental adipocytes, basal 2-deoxyglucose uptake was enhanced fourfold in clone 219 adipocytes, in accordance with increased expression of GLUT1 mRNA and protein. Whereas GLUT4 mRNA was similarly expressed in all of the adipocytes, GLUT4 protein appeared to decrease in clone 219 adipocytes. More importantly, subcellular fractionation studies showed that the localization of both GLUT1 and GLUT4 in the plasma membranes (PMs) was markedly increased in the basal state in clone 219 adipocytes compared with that in clone 233 and parental adipocytes, in which both glucose transporters were preferentially located in intracellular compartments. Consequently, insulin-induced translocation of GLUT1 was abolished in clone 219 adipocytes, although the remaining intracellular GLUT4 was still responsive to insulin stimulation, which led to the movement to the PM. As combined effects on the situation of GLUT1 and GLUT4, the foldness of insulin stimulation of glucose transport based on the basal activity was reduced in cells expressing constitutively active MAPKK. These results imply that chronic activation of MAPK could be one of the mechanisms for insulin resistance.

3T3 Cells↗
PubMed2001

[Production of HPV 16 major capsid protein L1 with baculovirus expression system].

BACKGROUND: To obtain human papillomavirus type 16(HPV 16) major capsid protein L1 with baculovirus expression system. METHODS: Using pFb1 as a vector,a recombinant baculovirus expressing plasmid,which contains HPV16L1 gene sequence,was generated. The constructed virus was infected into an insect cell line Sf9. RESULTS: After incubating at 27 degrees for 72 hours, the infected cells were collected and total cellular poteins were extracted. SDS-PAGE assay revealed a roughly 56 000 expressed protein and Western blot confirmed that the expressed protein arose from HPV16L1. CONCLUSIONS: HPV16 later protein L1 could be efficiently expressed with baculovirus expression system,and the expressed L1 protein remains to have good immunoreactivity. This study may supply a basic work for preparing virus-like particle and prophylactic HPV subunit vaccines.

Animals↗
PubMed1992

Tissue-specific expression of the skeletal alpha-actin gene involves sequences that can function independently of MyoD and Id.

The skeletal alpha-actin gene is a member of the sarcomeric contractile protein gene family and is specifically expressed in differentiated muscle. The skeletal alpha-actin gene is regulated efficiently by enhancer and regulatory sequences between nucleotide positions -1282 and -87. In the present study we have shown that the sequences 3' of nucleotide position -87 can functionally interact with the SV40 enhancer in a tissue-specific manner and can restrict the ubiquitous function of the SV40 enhancer to myogenic cells. Site-specific cassette mutagenesis was used to delimit the sequences upstream of the TATA motif (-32), between nucleotide positions -64 and -37, that mediate efficient expression in myogenic cells in the presence of the SV40 enhancer. The skeletal alpha-actin promoter was trans-activated by the helix-loop-helix (HLH) transcription factors MyoD, MRF-4, and Myogenin, in pluripotential 10T1/2 fibroblasts and trans-repressed by the HLH protein Id (inhibitor of differentiation) in myogenic C2C12 cells. This trans-regulation required sequences upstream of -87 and occurred independently of the two consensus E boxes (CANNTG) at positions +18 and +71. The -64/-37 region interacted with purified Sp1 and an unidentified protein(s), proximal regulatory factor(s) I (PRF-I). We conclude that the muscle-specific expression of the skeletal alpha-actin promoter is not simply determined by MyoD elements and enhancer and regulatory sequences, but that the minimal promoter contains important determinants of cell-specific transcription that can function independently of the helix-loop-helix transcription factors.

Actins↗
PubMed2003

[Vector construction for the intracellular domain of human receptor for advanced glycation end- products and expression of the fusion protein].

OBJECTIVE: To construct a eukaryotic expression vector for GST-tagged intracellular domain of human receptor for advanced glycation end-products (hRAGE), and to study the function of the expressed fusion protein and identify its interacting proteins. METHODS: The coding sequence of the intracellular fragment of hRAGE was amplified by PCR and inserted into pGEX-KG vector, a general GST fusion protein expression vector. After PCR identification, endonuclease digestion and DNA sequencing, the recombinant was transformed into E.coli BL21 to achieve the expression of GST fusion protein induced by isopropyl-beta-D-thiogalactoside (IPTG), followed by purification of the protein on glutathione-superflow resin. RESULTS: The recombinant of GST/hRAGE-C was constructed and identified by PCR, endonucleases digestion and DNA sequencing. After protein expression was achieved in E. coli, a molecular mass of 35 kD GST fusion protein was purified, whose molecular mass and purity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). CONCLUSION: The expression vector for intracellular domain of hRAGE has been successfully constructed and efficient expression of the fusion protein is obtained, which can be of value for further studies.

Genetic Vectors↗
PubMed2006

The uptake and fate of exogenous cellular DNA in mammalian cells.

Mammalian cells take up exogenous DNA very inefficiently. However, in the absence of viral vectors, DNA can be transfected into cells by co-precipitation with calcium phosphate and usually also with carrier DNA or by lipofection or electroporation. Such DNA can be expressed efficiently by cells. Alternatively, direct injection can also result in uptake and expression of transgenes. Without carefully designed means to target DNA specifically to integrate into the host genome, the vast majority of internalised DNA remains extra-chromosomal and is degraded. The likely fate of DNA which in low levels may contaminate vaccines derived from mammalian cell lines, will also be destruction. There is a theoretical risk of DNA integration events with random sequences of donor-derived DNA but the probability of that leading to serious adverse effects to the host is extremely small.

Administration, Oral↗
PubMed1998

Production of a hybrid protein consisting of the N-terminal fragment of urokinase and the C-terminal domain of urinary trypsin inhibitor in Escherichia coli.

We have constructed a hybrid protein (ATFHI) consisting of an N-terminal fragment from urokinase (ATF) and HI-8, which is the C-terminal domain of urinary trypsin inhibitor. The fusion genes for the hybrid proteins were engineered by PCR and cloned into expression plasmids. Under the control of the tac promoter, fusion genes were efficiently expressed in Escherichia coli. The hybrid proteins, produced as inclusion bodies in E. coli, were refolded by a dialysis method and purified by ion-exchange chromatography. ATFHI exhibited bifunctional activity related to antimetastatic effects: the urokinase receptor-binding activity of ATF and the inhibitory activity of HI-8 on plasmin.

Animals↗
PubMed1999

Truncated or chimeric endogenous protein antigens gain immunogenicity for B cells by stress protein-facilitated expression.

Truncated variants of the SV40 large T antigen (T-Ag) with an intact N terminus are as efficiently expressed in eukaryotic transfectants as wild-type (wt) T-Ag. Coprecipitation of N-terminal T-Ag fragments with the constitutively expressed, cytosolic stress protein hsp73 suggests that this chaperone stabilized expression of the truncated T-Ag fragments. In contrast to T-Ag, the 163-residue N-terminal preS domain of the hepatitis B surface antigen (HBsAg) is difficult to express. When the preS domain is C-terminally fused to a hsp73-binding cytoplasmic T-Ag (cT-Ag) fragment its stable expression as a chimeric cT-preS protein is obtained. DNA-based vaccination with plasmid DNA encoding either wt or hsp-associated mutant T-Ag elicited potent MHC class I-restricted, T-Ag-specific T cell responses. In contrast, DNA vaccination with hsp73-binding (mutant or chimeric) T-Ag variants, but not with wt T-Ag elicited T-Ag-specific antibody responses. Furthermore, vaccination with cT-preS-encoding plasmid DNA induced antibodies binding to the preS domain of the large HBsAg. Hence, hsp73-bound endogenous antigens efficiently stimulate antibody responses. These findings may be relevant for tumor immunology and autoimmunity.

Animals↗
PubMed2005

Glycogen synthase kinase 3beta functions to specify gene-specific, NF-kappaB-dependent transcription.

Loss of glycogen synthase kinase 3beta (GSK-3beta) in mice results in embryonic lethality via hepatocyte apoptosis. Consistent with this result, cells from these mice have diminished nuclear factor kappaB (NF-kappaB) activity, implying a functional role for GSK-3beta in regulating NF-kappaB. Here, we have explored mechanisms by which GSK-3beta may control NF-kappaB function. We show that cytokine-induced IkappaB kinase activity and subsequent phosphorylation of IkappaBalpha, p105, and p65 are not affected by the absence of GSK-3beta activity. Furthermore, nuclear accumulation of p65 following tumor necrosis factor treatment is unaffected by the loss of GSK-3beta. However, NF-kappaB DNA binding activity is reduced in GSK-3beta null cells and in cells treated with a pharmacological inhibitor of GSK-3. Expression of certain NF-kappaB-regulated genes, such as IkappaBalpha and macrophage inflammatory protein 2, is minimally affected by the absence of GSK-3beta. Conversely, we have identified a subset of NF-kappaB-regulated genes, including those for interleukin-6 and monocyte chemoattractant protein 1, that require GSK-3beta for efficient expression. We show that efficient localization of p65 to the promoter regions of the interleukin-6 and monocyte chemoattractant protein 1 genes following tumor necrosis factor alpha treatment requires GSK-3beta. Therefore, GSK-3beta has profound effects on transcription in a gene-specific manner through a mechanism involving control of promoter-specific recruitment of NF-kappaB.

Animals↗