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PubMed1996

In vitro and in vivo hepatoma cell-specific expression of a gene transferred with an adenoviral vector.

Recombinant adenoviruses are widely used for the transfer of foreign genes into various mammalian cells. However, the utilization of these vectors for cancer gene therapy requires the specific and efficient expression of the transferred gene in tumor cells. To obtain targeted expression in hepatoma cells, we constructed recombinant adenoviral vectors containing transcriptional elements from either the rat alpha-fetoprotein (AFP) or the human insulin-like growth factor II (IGFII) genes driving expression of the nuclear beta-galactosidase gene (nls lacZ). In vitro infection revealed that the AFP but not the IGFII transcriptional regulatory sequence controlled nls lacZ expression specifically in hepatoma cells. The same specificity was obtained in vivo in subcutaneous human hepatic tumors generated by engraftment of Huh7 hepatoma cells in nude mice as well as in primary liver tumors developed in rats and mice. No marker gene expression was detectable after AFP-nls lacZ gene transfer to normal rat liver parenchyma despite evidence for the presence of DNA encoding the nls lacZ gene. However, in vivo experiments with primary liver tumors in rats and mice also revealed that primary hepatoma cells were poorly infected by adenoviral vectors. Peritumoral and normal tissues were infected efficiently by adenoviral vectors. We conclude that hepatoma cell-specific expression of a transgene can be achieved with AFP regulatory sequences but that adenoviral vectors may not be the preferable vector for transferring genes in vivo in primary liver tumors.

Adenoviridae↗

PubMed1997

Delivery of a PCR amplified DNA fragment into cells: a model for using synthetic genes for gene therapy.

Synthetic genes offer many potential advantages over conventional plasmid DNA, such as simplicity in purification, absence of endotoxin contamination, and more importantly, flexibility in chemical modifications to render them specific properties. We have used PCR amplified fragments as a model to test the feasibility of using synthetic genes for gene therapy. The CAT reporter gene driven by the CMV promoter (CMV-CAT), ie a nuclear expression system, or by the bacteriophage T7 promoter (T7-CAT), ie a cytoplasmic expression system, was used to evaluate this concept. The expression efficiency of both plasmids (pUCCMV-CAT and pT7-CAT) and their corresponding PCR fragments (fCMV-CAT and fT7-CAT) were compared on a molar basis. Limited expression of CAT was found with fCMV-CAT. However, fT7-CAT consistently gave a CAT activity comparable to that of pT7-CAT. When fT7-CAT was codelivered with pCMV/T7-T7pol (a self-amplifying-T7 RNA polymerase autogene), high CAT activity could be detected up to 9 days. This expression was much longer than the duration of expression with a nuclear expression system. These encouraging results imply that gene therapy with synthetic genes could be both feasible and efficient.

Gene Expression↗

PubMed2000

Gene transfer into the fetal primate: evidence for the secretion of transgene product.

In utero adenoviral-mediated transfer of genes via the amniotic fluid results in sustained high-efficiency expression in rodent lung and intestine. Rhesus macaque (Macaca mulatta) fetuses were injected with adenovirus vectors encoding reporter genes at different gestational ages to evaluate feasibility and timing in primates. The fetuses developed normally following gene transfer and no maternal adverse affects were noted. Highly efficient viral uptake and transgene protein expression occurred in the target organs. The lungs exhibited no immune response and transgenic protein was observed up to 30 days postinfection. Unexpectedly, large amounts of reporter gene protein were released, apparently from the lung, into the circulation and accumulated in the renal proximal tubules and bladder. PCR detection for adenovirus DNA was consistently negative in tissues not in contact with the amniotic fluid, such as kidneys, liver, gonads, and eyes. Treatment of primate fetuses at 110 days gestation with an adenovirus expressing the cystic fibrosis transmembrane conductance regulator (cftr) gene resulted in accelerated differentiation of the lung. These studies demonstrate the efficacy of in utero gene therapy in primates and its potential application to genetic diseases.

Adenoviridae↗

PubMed1999

Muscle-mediated gene therapy.

Transfer of therapeutic genes into muscle tissue holds promise for the treatment of a variety of muscular dystrophies, serum protein deficiencies and vascular proliferative disorders. Recent progress achieved in development of improved vectors allowed prolonged and efficient expression of genes encoding therapeutic proteins in muscle cells. The most important advances include: novel plasmid DNA vectors and methods for their efficient transfection in vivo, helper-dependent adenoviral vectors, allowing long-term gene expression and effective readministration in immunocompetent hosts and adeno-associated vectors. On the other hand, recent progress in this field has been facilitated by development of systems that enable regulated therapeutic gene expression in muscle tissue.

Animals↗

PubMed1991

Multiple sets of adjacent mu E1 and oct-1 binding sites upstream of the pseudorabies virus immediate-early gene promoter.

Binding of cellular proteins to specific motifs in the promoter region of the immediate-early gene of pseudorabies virus was studied. The region was dissected into several portions that were used with HeLa cell nuclear proteins in mobility shift assays, DNase I footprinting, and a methylation interference assay. Close to the transcription start site (nucleotide + 1) are a TATA-box (-26 to -29), an Sp 1-binding motif (GGGGCGGGC) (-45 to -54), a CCAAT motif (-66 to -70), and, further upstream, an NF-microE1-binding site (AAGATGGC) (-161 to -168). Binding of a protein to the Sp1 site was demonstrated. Competition experiments show that the CCAAT motif might bind the NF-microE1 factor, rather than any of the known CCAAT-specific factors. Four domains were identified further upstream (nucleotides -200 to -500) from this promoter, each of which contained closely associated motifs where cellular transcription factors NF-microE1 and oct-1 could bind. These domains comprise what we call the upstream element. The orientation of the four NF-microE1 motifs in the upstream element is opposite to the orientation of the two NF-microE1 motifs located closer to the transcription start site. Transient expression of reporter genes was used to study the activity of the upstream element after transfection into 3T3 and HeLa cells. The upstream element was necessary for efficient expression of the pseudorabies virus immediate-early gene and increased somewhat the efficiency of the herpes simplex virus thymidine kinase promoter.

Base Sequence↗

PubMed2003

Expression of a novel regenerating gene product, Reg IV, by high density fermentation in Pichia pastoris: production, purification, and characterization.

Human regenerating (Reg) gene products are regionally expressed by gut-derived tissues, and are markedly up-regulated in cancer and in diseases characterized by mucosal injury. We recently identified Reg IV, a novel regenerating gene product that is uniquely expressed by the normal distal gastrointestinal mucosa. The function remains poorly understood due to the lack of significant purified Reg IV for biochemical and functional studies. Recombinant human Reg IV was efficiently expressed under the control of the AOX1 gene promoter in Pichia pastoris using the MutS strain KM71H. We describe the unique conditions that are required for efficient production of Reg IV protein in high density fermentation. Optimal protein expression was obtained by reduction of the fermentation temperature and addition of casamino acids as a supplemental nitrogen source and to minimize the activity of yeast produced proteases. Recombinant Reg IV protein was purified by tangential flow filtration and reverse phase chromatography. The purified protein was characterized by amino terminus sequence analysis and MALDI-TOFMS showing that the engineered protein had the expected sequence and molecular weight without secondary modification. Recombinant Reg IV was further characterized by specific monoclonal and polyclonal reagents that function for Western blot analysis and for immunolocalization studies.

Amino Acid Sequence↗

PubMed1994

Expression in Escherichia coli and phosphorylation with cAMP-dependent protein kinase of the N-terminal region of human endothelial actin-binding protein.

The N-terminal region of human endothelial actin-binding protein was subcloned and expressed in the pT 7-7/E. coli BL 21 (DE 3) system. This peptide was efficiently expressed in E. coli as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by western analysis using a monoclonal antibody raised against this part of the molecule. As predicted by the amino acid sequence this truncated protein (residues 21-1569), corresponding to 164 KD and containing the actin-binding domain near the amino-terminus, could be phosphorylated by cAMP-dependent protein kinase unmasking a phosphorylation site which is not apparent in the native ABP protein.

Actins↗

PubMed1991

Bacterial expression of rat liver succinyl-CoA synthetase alpha-subunit. Factors that contribute to blocked translation of transcripts encoding a mitochondrial signal sequence.

This study comprises a detailed evaluation of factors that are necessary to achieve high levels of expression of eukaryotic proteins in bacterial systems. We attempted to express a rat liver cDNA clone encoding the precursor to the alpha-subunit of succinyl-CoA synthetase in an Escherichia coli expression system, without success. Removal of the region encoding the mitochondrial signal peptide (115 nucleotides) allowed efficient expression of the mature protein. This nucleotide sequence was shown to block expression at the level of translation. Two regions within this fragment were able to block the expression of other genes such as E. coli lacZ. Inhibition of expression was due to the close proximity of these inhibitory sequences with the translation initiation region (TIR). Insertion of a spacer between the inhibitory sequence and the TIR relieved the block in translation. Analysis of the 115-nucleotide fragment identified sequences capable of extensive base-pairing with the Shine-Dalgarno and surrounding sequences. Such secondary structures are capable of blocking the formation of competent translation initiation complexes.

Animals↗

PubMed1985

The mom gene of bacteriophage mu: a unique regulatory scheme to control a lethal function.

The mom gene of bacteriophage Mu encodes a DNA modification function which converts adenine to acetamido adenine in a sequence-specific manner. The mom gene itself is subject to a complex regulation: gene expression requires methylation by the Escherichia coli Dam methylase of specific sites upstream of the mom promoter and transactivation of the promoter by a Mu gene product. The requirement for transactivation can be overcome when mom is transcribed from foreign promoters. When cloned into various sites in pBR322, the mom gene is always found in an orientation where transcription from vector promoters is excluded. The productive orientation is lethal to the cell. This effect is mediated by the concerted action of the mom gene product and the product of gene com (control of mom, previously termed ORF-x) whose coding region overlaps the 5-coding region of the mom gene. When mom is expressed from its own promoter, internal deletions in com completely abolish expression of the mom gene. Fragments lacking the 5' end of com can be cloned downstream of constitutive plasmid promoters. The com gene product itself is not lethal to the cell. The region encoding mom has been cloned in pL expression vectors. The mom gene product, a peptide of 27 kDal, has been visualized on gels. Efficient expression of Mom from pL requires gene com. A fusion between MS-2 polymerase and com has been generated. The fusion product is made in large amounts, whereas the mom gene product is not overproduced although the gene is present on the same transcriptional unit.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage mu↗

PubMed2004

Production of a biologically active growth hormone from giant catfish (Pangasianodon gigas) in Escherichia coli.

Giant catfish growth hormone (gcGH) cDNA was cloned and expressed in E. coli. The expected 20.5 kDa protein corresponded to the mature gcGH and was efficiently expressed. This protein was produced as inclusion bodies and comprised about 20% of total cellular proteins. The recombinant hormone promoted growth when injected intramuscularly or intraperitoneally into goldfish (Carassius auratus) at 0.1 or 1 microg soluble gcGH per g fish body wt per week. In addition, the recombinant gcGH inclusions had growth-promoting activity similar to that of the soluble form when the fish was received either by intraperitoneal injection or by oral administration.

Animals↗

PubMed2007

Functional analysis of a cotton glucuronosyltransferase promoter in transgenic tobaccos.

The 5' fragment (1 647 bp) of the cotton glucuronosyltransferase gene (GhGlcAT1) was transcriptionally fused to the beta-glucuronidase (GUS) gene, and functionally analyzed for important regulatory regions controlling gene expression in transgenic tobacco plants. GUS activity analysis revealed that the full-length promoter drives efficient expression of the GUS gene in the root cap, seed coat, pollen grains and trichomes. Exposure of the transgenic tobacco to various abiotic stresses showed that the promoter was mainly responsive to the sugars (glucose and sucrose) as well as gibberellic acid. Progressive upstream deletion analyses of the promoter showed that the region from -281 to +30 bp is sufficient to drive strong GUS expression in the trichomes of shoot, suggesting that the 311 bp region contains all cis-elements needed for trichome-specific expression. Furthermore, deletion analysis also revealed that the essential cis-element(s) for sucrose induction might be located between -635 and -281 bp. In addition, sequence analysis of the regulatory region indicated several conserved motifs among which some were shared with previously reported seed-specific elements and sugar-responsive elements, while others were related with trichome expression. These findings indicate that a 1 647-bp fragment of the cotton GhGlcAT1 promoter contains specific transcription regulatory elements, and provide clues about the roles of GhGlcAT1 in cotton fiber development. Further analyses of these elements will help to elucidate the molecular mechanisms regulating the expression of the GhGlcAT1 gene during fiber elongation.

Base Sequence↗

PubMed2001

Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression.

Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone-like protein H-NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37 degrees C (e.g. 40 degrees C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37 degrees C (e.g. 25 degrees C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H-NS-mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H-NS-nucleoprotein complex.

Amino Acid Sequence↗

PubMed2002

Dual control of subtilin biosynthesis and immunity in Bacillus subtilis.

The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and beta-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. beta-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.

Anti-Bacterial Agents↗

PubMed2004

Rabies virus nucleoprotein as a carrier for foreign antigens.

Rabies virus (RV) nucleoprotein (N) tightly encapsidates the genomic and antigenomic RNA of RV to form the viral ribonucleoprotein (RNP) complex. Antigens, such as N, presented in a highly organized structure are sufficient and even desirable to activate B cells to proliferate and produce antibodies. In addition to activating B cells to proliferate, it has been shown that RV N in the RNP complex induces potent T helper cell responses resulting in long-lasting and strong humoral immune responses against RV. The possibility to systematically incorporate foreign genes into the genome of RV and produce a recombinant virus allows us to examine whether the immunogenicity of foreign antigens can be enhanced by incorporation into the RV RNP structure. To test this hypothesis we constructed a recombinant RV expressing a RV N-GFP fusion protein. The chimeric N-GFP fusion protein was efficiently expressed and incorporated into RV RNP and virions. Moreover, the recombinant RNP induces a strong humoral immune response against GFP in mice. In contrast, mice inoculated with GFP alone or a combination of wild-type RV RNPs and GFP did not trigger any GFP-specific humoral responses using the same immunization schedule. These data indicate the usefulness of RV-based vectors as killed vaccines against other infectious diseases.

Animals↗

PubMed2001

Identification of a trafficking determinant localized to the Kv1 potassium channel pore.

The repertoire of Kv1 potassium channels expressed in presynaptic terminals of mammalian central neurons is shaped by intrinsic trafficking signals that determine surface-expression efficiencies of homomeric and heteromeric Kv1 channel complexes. Here, we show that a determinant controlling surface expression of Kv1 channels is localized to the highly conserved pore region. Point-mutation analysis revealed two residues as critical for channel trafficking, one in the extracellular "turret" domain and one in the region distal to the selectivity filter. Interestingly, these same residues also form the binding sites for polypeptide neurotoxins. Our findings demonstrate a previously uncharacterized function for the channel-pore domain as a regulator of channel trafficking.

Amino Acid Sequence↗

PubMed1989

Expression of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase and its kinase domain in Escherichia coli.

The rat liver bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (ATP:D-fructose-6-phosphate 2-phosphotransferase/D-fructose-2,6-bisphosphate 2-phosphohydrolase, EC 2.7.1.105/EC 3.1.3.46) and its separate kinase domain were expressed in Escherichia coli by using an expression system based on bacteriophage T7 RNA polymerase. The bifunctional enzyme (470 residues per subunit) was efficiently expressed as a protein that starts with the initiator methionine residue and ends at the carboxyl-terminal tyrosine residue. The expressed protein was purified to homogeneity by anion exchange and Blue Sepharose chromatography and had kinetic and physical properties similar to the purified rat liver enzyme, including its behavior as a dimer during gel filtration, activation of the kinase by phosphate and inhibition by alpha-glycerol phosphate, and mediation of the bisphosphatase reaction by a phosphoenzyme intermediate. The expressed 6-phosphofructo-2-kinase also started with the initiator methionine but ended at residue 257. The partially purified kinase domain was catalytically active, had reduced affinities for ATP and fructose 6-phosphate compared with the kinase of the bifunctional enzyme, and had no fructose-2,6-bisphosphatase activity. The kinase domain also behaved as an oligomeric protein during gel filtration. The expression of an active kinase domain and the previous demonstration of an actively expressed bisphosphatase domain provide strong support for the hypothesis that the hepatic enzyme consists of two independent catalytic domains encoded by a fused gene.

Animals↗

PubMed1992

A simple method for overproduction and purification of p24 Gag protein of human immunodeficiency virus type 1.

A simple method for the overproduction in Escherichia coli and purification of major core protein p24 of human immunodeficiency virus type 1 (HIV-1) was described. The gag-pol region encoding p24, p15, and protease was fused to 3' end of lacZ gene on plasmid. A LacZ-Gag fusion protein, the major primary product, is designed to be cleaved by the HIV-1 protease coexpressed through frameshifting. In fact, p24 and its immediate precursor, p25, were produced in the cells grown at 25C, but not at 37C. When the gag and pol frames were fused in-frame to express the protease without frameshifting, the main product, a LacZ-Gag-Pol fusion protein, was efficiently processed to give p24 exclusively both at 37C and 25C, suggesting more efficient expression of the protease. Recombinant p24 was purified to near homogeneity by a simple three-step procedure. The amino-terminal sequence of the recombinant p24 was the same as that of p24 deduced from nucleotide sequence, indicating that correct processing occurred in E. coli by the coexpressed protease. The method described here provides a means to obtain a large amount of highly pure p24, which is useful for crystallographic and functional studies, preparation of specific antibody, and diagnostic and prognostic uses.

Chromobox Protein Homolog 5↗

PubMed1994

A baculovirus gene involved in late gene expression predicts a large polypeptide with a conserved motif of RNA polymerases.

We have identified and sequenced a novel baculovirus gene, late expression factor eight gene (lef-8), of Autographa californica nuclear polyhedrosis virus that is necessary for efficient expression from late and very late virus gene promoters in a transient expression assay. The predicted gene product, LEF-8, has a molecular mass of 102 kDa and contains a conserved sequence motif, GXKX4HGQ/NKG, found in DNA-directed RNA polymerases throughout the animal, plant, and microbial kingdoms.

Amino Acid Sequence↗