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At least 1,531 records · Page 85Linked to original sources
PubMed1990

Cloning and expression of the 58 kd beta subunit of the inhibitory glycine receptor.

The inhibitory glycine receptor (GlyR) mediates post-synaptic inhibition in spinal cord and other regions of the CNS. Purified mammalian GlyR contains two membrane-spanning subunits 48 kd (alpha) and 58 kd (beta) plus a 93 kd receptor-associated cytoplasmic protein. Here, the primary structure of the beta subunit was deduced from cDNAs isolated from rat spinal cord and brain cDNA libraries. The predicted amino acid sequence exhibits 47% identity to the previously characterized rat alpha 1 polypeptide. Northern blot analysis revealed high levels of beta subunit transcripts in postnatal spinal cord, cerebellum, and cortex. Nuclear injection into Xenopus oocytes of a beta subunit cDNA engineered for efficient expression generated weak glycine-activated chloride currents that were insensitive to the classic GlyR antagonist, strychnine. Our data indicate a differential expression of GlyR alpha and beta subunits in the rat nervous system and support a structural role of the beta polypeptide in the native receptor complex.

Amino Acid Sequence↗
PubMed2005

Expression of spider flagelliform silk protein in Bombyx mori cell line by a novel Bac-to-Bac/BmNPV baculovirus expression system.

Bombyx mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system (BES) has a lot of advantages such as high expression efficiency, convenience, and low feeding cost. In this report, we used a recently developed BmNPV bacmid, which could infect both B. mori cell lines and silkworm larvae. The results showed it takes only 7 to 10 days to generate recombinant baculovirus and permit the rapid isolation from small-scale cultures and then use it to transfect B. mori cell lines, compared to traditional homologous recombination method, which needs at least 40 days for multiple rounds of purification and amplification of viruses. Using this BES, we expressed a recombinant spider flagelliform protein in BmN cell line, which was around 37 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. The BmNPV bacmid system using silkworm would be very attractive for expression of target proteins.

Amino Acid Sequence↗
PubMed1990

Control and expression of 3' open reading frames in clover yellow mosaic virus.

The genomic RNA of clover yellow mosaic virus (CYMV) contains at least seven open reading frames (ORFs) which are organized in a more elaborate array than in other sequenced members of the potexvirus group. We have investigated the strategy by which ORFs located in the 3' region of CYMV's genomic RNA are differentially expressed by correlating the location of the 5' termini of the two abundant viral subgenomic RNAs with their coding potential. We have mapped the 5' termini of the subgenomic RNAs precisely to the nucleotide level and have shown that both are capped. The larger 2.1-kb subgenomic RNA encodes as its 5' ORF a 25-kDa polypeptide, whose function is unknown. The smaller 1.0-kb subgenomic RNA can encode only the 23-kDa coat protein. All four ORFs in the 3' 1095 residues of CYMV are efficiently expressed in vitro, but of these only coat protein, which can be expressed from a subgenomic RNA, is detectable in CYMV infected tissue. For this reason, we believe that expression of ORFs in the 3' one-third of CYMV RNA are controlled at the transcriptional level.

Base Sequence↗
PubMed1991

Identification of a novel transcription factor, ACF, in cultured avian fibroblast cells that interacts with a Marek's disease virus late gene promoter.

Interactions between factors in duck and chick embryo fibroblast (DEF and CEF, respectively) nuclear extracts and the Marek's disease virus (MDV) gp57-65 gene promoter were investigated. Results of in vitro transcription and gel mobility-shift assays indicated that multiple cellular factors interact with 5'-flanking sequences of the MDV gp57-65 gene. One sequence-specific DNA binding activity (termed ACF for avian cell factor(s)) was identified by interaction of DEF and CEF nuclear extract proteins with a particular site (nucleotides -193 to -177) in the MDV gp57-65 gene promoter. Binding of ACF to its apparent recognition sequence, contained within the 17-bp oligonucleotide 5'-CTAGTTTACTTGTTTGT-3' (ACF-12), was highly sequence-specific. Radiolabeled ACF-12 oligonucleotide bound significant ACF protein in the presence of a 400-fold molar excess of unlabeled nonspecific competitor DNA. A similar amount of specific competitor completely abolished ACF binding to probe DNA. Deletion of the ACF binding site from MDV gp57-65 gene promoters linked to a chloramphenicol acetyltransferase (CAT) reporter gene reduced expression of CAT activity by twofold relative to that seen with a gp57-65 promoter-CAT construct containing an intact ACF binding site. Transfection inhibition assays using double-stranded ACF binding site competitors reduced steady-state levels of gp57-65 mRNA in MDV infected cells by over twofold relative to those in control infected cells. Introduction of a similar amount of nonspecific double-stranded oligonucleotide had no adverse effect on gp57-65 mRNA levels. These data suggest that ACF is important for efficient expression of gp57-65.

Animals↗
PubMed2002

Lipofection of early passages of cell cultures derived from murine adenocarcinomas: in vitro and ex vivo testing of the thymidine kinase/ganciclovir system.

Early passages of cultured cells derived from four spontaneous Balb/c murine adenocarcinomas were used to explore the feasibility of a nonviral HSVtk-based suicide gene therapy system. After lipofection with pCMVtk, the transiently HSVtk expressing P07 (lung), M3, M05, and M38 (mammary gland) cells were, respectively, about 130-, 30-, 120-, and 170-fold more sensitive to ganciclovir (GCV) in vitro than their respective controls. Eighty percent of Balb/c mice subcutaneously inoculated with ex vivo pCMVtk-lipofected P07 cells, followed by intraperitoneal GCV injection for 7 days, displayed a complete inhibition of tumor growth for over 70 days. Control animals started to display tumors 13 days after inoculation. We present evidence showing that early passages of cultured tumor cells can efficiently express lipofected genes and that they are sensitive to the lipoplex-mediated HSVtk/GCV system.

Adenocarcinoma↗
PubMed1995

The transcriptional regulation of human aldehyde dehydrogenase I gene. The structural and functional analysis of the promoter.

Human cytosolic aldehyde dehydrogenase 1 (ALDH1) plays a role in the biosynthesis of retinoic acid that is a modulator for gene expression and cell differentiation. Northern blot analysis showed that liver tissue, pancreas tissue, hepatoma cells, and genital skin fibroblast cells expressed high levels of ALDH1. Sequence analysis showed that the 5'-flanking region contains a number of putative regulatory elements, such as NF-IL6, HNF-5, GATA binding sites, and putative response elements for interleukin-6, phenobarbital and androgen, in addition to a noncanonical TATA box (ATAAA) and a CCAAT box. Functional characterization of the 5'-regulatory region of the human ALDH1 gene was carried out by a fusion to the chloramphenicol acetyltransferase gene. A construct containing 2.6 kilobase pairs of the 5'-flanking region was efficiently expressed in hepatoma Hep3B cells, but not in erythroleukemic K562 cells or in fibroblast LTK- cells, which do not express ALDH1. Within this region, we define a minimal promoter (-91 to +53) that contains positive regulatory elements. The study using site-directed mutagenesis demonstrated that the CCAAT box region is the major cis-acting element involved in basal ALDH1 promoter activity in Hep3B cells. Gel mobility shift assays showed that NF-Y and other octamer factors bound CCAAT box and an octamer motif sequence, but not GATA site existing in the minimal promoter region. Two additional DNA binding activities associated with the minimal promoter were found in the nuclear extract from Hep3B cells, but not from K562 cells. These results offer the possible molecular mechanism of the cell type-specific expression of ALDH1 gene.

Aldehyde Dehydrogenase↗
PubMed1998

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

Adenoviridae↗
PubMed2006

Regulation of matrix metalloproteinase production and tumor cell invasion by four monoclonal antibodies against different epitopes of HAb18G/CD147 extracellular domain.

HAb18G/CD147, a membrane spanning molecule and highly expressed in hepatocellular carcinoma (HCC) cells, was shown to stimulate the production of matrix metalloproteinases (MMPs) in the interaction of tumor cells and fibroblasts. Studies on the EMMPRIN/CD147 showed that CD147 extracellular domain is involved in the induction of MMPs. To study the biological molecular function of HAb18G/CD147 extracellular domain (HAb18G/CD147-ED) on production of MMPs following mediated tumor cell invasion, we isolated four novel monoclonal anibodies (MAbs)-1B3, 3B3, HAb18Gedomab1, and HAb18Gedomab2-against HAb18G/CD147-ED by immunization of BALB/c mice with purified HAb18G/CD147-ED fragments, which were efficiently expressed in Escherichia coli. Gelatin zymography and Boyden chamber assays were used to identify the production of MMPs in the co-cultured human fibroblast and HCC cells, and to quantify the migrated cells in the presence of the generated MAbs. The results showed that two MAbs (1B3 and 3B3) inhibited [corrected] the secretion of MMP-2 and [corrected] the HCC cell invasion, whereas the other two MAbs (HAb18Gedomab1 and HAb18Gedomab2) had reverse function [corrected] FCM additive assay showed that four MAbs recognized different epitopes of HAb18G/CD147-ED. Taken together, the results suggest that various regions of HAb18G/CD147-ED participated in the regulation of MMP secretion.

Animals↗
PubMed1995

mRNA expression of vimentin gene in lens of transgenic mouse and DNA amplification in human cataracts.

PURPOSE: To investigate the role of vimentin gene in cataractogenesis. METHODS: The 12.7kb chicken vimentin genes were microinjected into the male pronuclei of 918 fertilized mice eggs. 841 injected embryos were transferred into oviducts of pseudopregnant recipient females, of which 12 pregnant mice gave birth to 49 offspring mice. The integration and expression of exogenous gene in the offspring were analysed by Southern and Northern blot hybridizations. In the human senile cataract, the lens vimentin gene was analyzed with the chicken vimentin gene probe. RESULTS: It showed that four of F1 offspring were transgenic mice in which the chicken vimentin gene was integrated in their genomes. The transgenic band was 12kb, similar to the 12.7kb chicken vimentin fragment injected. One 2kb vimentin mRNA was visualized on E2 mouse lens blot, which revealed that the chicken vimentin gene was efficiently expressed in this transgenic mouse. In the human senile cataract lens, 12kb BamHI-restricted vimentin fragments displayed a stronger hybridization signal than that of the control lens in Southern blot analysis. It implies that the formation of human senile cataract may be associated with the amplification of vimentin gene. CONCLUSIONS: We have successfully developed four transgenic mice bearing chicken vimentin gene and having mRNA expression which can be used for further study. It is to be observed if the normal lens cell function is affected by the expressed product and cataract occurs in our transgenic mice. The cause of the gene amplification in human cataract remains for further investigation.

Adult↗
PubMed1985

Stable transfer and restricted expression of a cloned class I gene encoding a secreted transplantation-like antigen.

The identification of a unique major histocompatibility complex class I gene, designated Q10, which encodes a secreted rather than a cell surface antigen has led to questions regarding its potential role in regulating immunological functions. Since the Q10 gene is specifically activated only in the liver, we sought to define the molecular mechanisms which control its expression in a tissue-specific fashion. Results obtained by transfection of the cloned Q10 gene, either in the absence or presence of a heterologous transcriptional enhancer, into a variety of cell types of different tissue derivations are consistent with the Q10 gene being regulated at two levels. The first is by a cis-dependent mechanism which appears to involve site-specific DNA methylation. The second is by a trans-acting mechanism which would include the possibility of an enhancer binding factor. The ability to efficiently express the Q10 gene in certain transfected cell lines offers an opportunity to obtain this secreted class I antigen in quantities sufficient for functional studies; this should also make it possible to define regulatory sequences which may be responsible for the tissue-specific expression of Q10.

Animals↗
PubMed1986

Characterization of 5'-flanking region of heart myosin light chain 2A gene. Structural and functional evidence for promoter activity.

Two recombinant clones, lambda LC5 and lambda LC13, encompassing the entire regulatory myosin light chain 2 (MLC2A) gene of chicken heart muscle were isolated. Of these, lambda LC5 which contains a large 5'-flanking sequence of about 7.0 kb, was characterized by a partial nucleotide sequence analysis. A TATA-like sequence (TATTTTTA) and a CAAT-box (CAAAAGT) are located at positions -32 and -59, respectively, which most likely constitute the functional promoter region in the gene. Based on primer extension reaction with a synthetic 20-mer corresponding to the 5'-leader sequence and total poly(A+) RNA, the probable transcription initiation site in the gene was located. The gene promoter activity was demonstrated following transient expression of recombinant genomes containing the chicken upstream sequence fused to the bacterial chloramphenicol acetyltransferase (CAT) or to the rat preproinsulin II genes. The extracts from a Quail fibroblast cell line (QT35) transfected with the construct (pLCo5.2iCat) containing the putative chicken promoter, and the CAT gene promoted the formation of 3'-acetate chloramphenicol. Another construct (pBC12LC5.2f) contains the rat preproinsulin II gene placed under the control of chicken promoter and a simian virus 40 origin of replication. Transfection of COS cell line with pBC12LC5.2f DNA resulted in an efficient expression of rat preproinsulin mRNA initiating from the chicken promoter. The transfection assay also allowed detection of chicken MLC2A gene transcripts by S1-nuclease protection of end-labeled DNA probes. A comparison of the MLC2A upstream gene sequence with those available for skeletal myosin light chains revealed no common sequence elements, suggesting that cardiac MLC2A gene promoter region has diverged considerably from its counterparts in skeletal muscle.

Amino Acid Sequence↗
PubMed1986

Expression of the E. coli O6-methylguanine-methylphosphotriester methyltransferase gene in mammalian cells.

Many prokaryotic and eukaryotic cells contain enzymes that repair damage introduced into their DNA following exposure to chemical, physical and biological agents. One such lesion that has received considerable attention is the potentially miscoding and mutagenic base O6-alkylguanine which is produced in varying amounts in DNA following reaction with monofunctional alkylating agents. As part of a study to assess the role of this lesion and its repair in the processes of cytotoxicity, mutagenicity and transformation, we have recently cloned the Escherichia coli gene which codes for the protein responsible for the repair of such damage in DNA. In the present study we describe the construction of a plasmid which allows the efficient expression of the bacterial gene in mammalian cells.

Animals↗
PubMed1986

The alpha 2 cDNA sequence of human haptoglobin carries a bacterial promoter functional in vivo.

Various constructions of human haptoglobin (Hp) cDNA coding either for the complete alpha 2FS beta precursor protein or only for the beta subunit have been placed under the control of the lambda PR promoter in the bacterial expression vector pCQV2 (Queen, 1983). In addition to the expected 45,000 dalton polypeptide synthesized after induction of the PR promoter, the complete alpha 2FS beta constructions constitutively express a smaller polypeptide of approximately 30,000 dalton corresponding to a truncated Hp protein. Computer analysis of the HpcDNA revealed the presence of two strong potential bacterial promoters (alpha 2 PF and alpha 2 PS) located in the duplicated alpha 2FS sequence. Both Hp promoter signals are followed by potential mRNA start sites and ribosome binding sites at a compatible distance from initiation codons. In addition, the Hp alpha 2 cDNA sequence, when fused upstream to the cDNA coding for alpha 1-antitrypsin, constitutively promotes in vivo the efficient expression of an hybrid protein specifically recognized by antibodies raised against alpha 1-antitrypsin or haptoglobin.

Cloning, Molecular↗
PubMed1988

Insertional activation of a promoterless thymidine kinase gene.

A plasmid carrying a promoterless herpes simplex virus thymidine kinase gene was transfected via calcium phosphate precipitation into LM (tk-) mouse fibroblast cells. The transfected gene was efficiently expressed, as the transfected cells grew perfectly well in selective hypoxanthine-aminopterin-thymidine medium, suggesting that the thymidine kinase-coding region became linked to a promoterlike element on integration into the recipient genome. To investigate the structure of the surrogate promoter, we first isolated the integrated gene from a genomic library. The nucleotide sequence of the DNA adjacent to the thymidine kinase-coding sequence was then determined. We found, first, that the integration of the transfected DNA apparently occurred by a blunt end ligation mechanism involving no obvious sequence similarities between integrated and recipient DNA and, second, that the 5'-flanking region included a TATA box, two CCAAT boxes, and a GC box element. However, the TATA box motif and the most proximal CCAAT box appeared to be sufficient for full promoter activity, as determined by the transfection efficiencies of appropriate plasmid constructs. Except for these canonical promoter elements, the surrogate promoter had no obvious similarities to known thymidine kinase gene promoters.

Animals↗
PubMed2000

Ultrasound facilitates transduction of naked plasmid DNA into colon carcinoma cells in vitro and in vivo.

One approach to improve the efficacy of in vivo gene therapy, with the aim at enhancing expression of the transgene, involves utilization of mechanical forces to facilitate transduction of DNA into cells. In this study, we evaluated the feasibility of mechanical insonation in gene transfers with naked DNA plasmid loading both in vitro and in vivo. We used an ultrasound probe, which can focus the ultrasonic beam in the exit zone of the probe. The reporter pcDNA3-lacZ plasmid, containing Escherichia coli lacZ or the beta-galactosidase gene (beta-gal), and the neomycin 3'-phosphotransferase gene (neo), was used for evaluation of transfer efficiency. Expression of beta-gal in MC38 murine colon carcinoma cells was measured after insonation of 20 W/cm2 with continuous 1.0-MHz wave exposure. In a transient assay, significant numbers of cells were transduced with the beta-galactosidase gene. After cells were treated with geneticin, we also observed a difference in colonogenicity between noninsonated and insonated groups. When MC38 cells were implanted in syngeneic mice and plasmid was injected, the insonation that followed facilitated beta-galactosidase expression. These results indicate that insonation represents a potential approach for gene therapy when combined with naked DNA plasmid injection.

Adenocarcinoma↗
PubMed1990

Temperature elevation enhances cell surface expression of measles virus fusion protein in infected cells.

Cell fusion proceeded gradually in measles virus-infected cells incubated at 35 degrees C. Shift-up of incubation temperature to 39 degrees C induced rapidly increased cell fusion in spite of the cessation of de novo synthesis of the fusion (F) protein. Pulse-chase experiments showed that there was little difference in the acquisition of immunoreactivity by haemagglutinin (H) and F proteins between the two temperatures. H protein was detected on the cell surface 60 min after the chase at either temperature. However, appearance of F protein on the cell surface took less than 3 h at 39 degrees C whereas it took 5 h at 35 degrees C. These data indicate that temperature elevation induces more efficient expression of F protein on the cell surface accompanied by marked syncytium formation in measles virus-infected cells.

Cell Membrane↗
PubMed2006

Expression of CD83 is regulated by HuR via a novel cis-active coding region RNA element.

Dendritic cells are the most potent of the antigen-presenting cells and are characterized by surface expression of CD83. Here, we show that the coding region of CD83 mRNA contains a novel cis-acting structured RNA element that binds to HuR, a member of the ELAV family of AU-rich element RNA-binding proteins. Transient transfection of mammalian cells demonstrated that this CD83 mRNA-derived element acts as a post-transcriptional regulatory element in cells overexpressing HuR. Notably, binding of HuR to the CD83 post-transcriptional regulatory element did not affect mRNA stability. Using RNA interference, we show that HuR mediated efficient expression of CD83. In particular, HuR was required for cytoplasmic accumulation of CD83 transcripts. Likewise, inhibition of the CRM1 nuclear export pathway by leptomycin B or overexpression of a defective form of the nucleoporin Nup214/CAN diminished cytoplasmic CD83 mRNA levels. In summary, the data presented demonstrate that the HuR-CRM1 axis affects the nucleocytoplasmic translocation of CD83 mRNA under regular physiological conditions.

Active Transport, Cell Nucleus↗
PubMed1983

Cloning, sequencing, and secretion of Bacillus amyloliquefaciens subtilisin in Bacillus subtilis.

The subtilisin gene from B. amyloliquefaciens has been cloned and expressed under its own promoter on a high copy plasmid, pBS42, in Bacillus subtilis I-168 (Marburg strain). Greater than 95 percent of the expressed protease activity is secreted, and the activity is sensitive to inhibition by phenylmethylsulfonyl fluoride as expected for subtilisin. Bacillus subtilis transformants carrying the Bacillus amyloliquefaciens subtilisin gene in pBS42 (called pS4) secreted large amounts of a protein not seen in control pBS42 transformants. This protein migrated in SDS gels near the position of authentic subtilisin. The complete nucleotide sequence of the cloned gene has been determined using dideoxy sequencing methods. Ba131 exonuclease digestion studies at the 5' end of the gene have defined a 31 base pair stretch necessary for efficient expression of subtilisin. In addition to this putative promoter region, sequences have been assigned for ribosome binding, translation initiation, a signal peptide, the mature enzyme, and translation and transcription termination. A most interesting feature of the gene is a sequence of unknown function coding for roughly 75 amino acids between the signal sequence and the mature enzyme. It is proposed that this region serve as a pro-peptide as is commonly found in eukaryotic secreted proteases.

Amino Acid Sequence↗