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At least 973 records · Page 54Linked to original sources
PubMed1998

Differential gene expression in Arabidopsis monitored using cDNA arrays.

Large numbers of genes are being discovered on a daily basis for a variety of organisms including Arabidopsis thaliana. To obtain more functional information on these genes, efficient expression monitoring methods need to be developed. In this report we studied the steady-state mRNA levels of over 800 Arabidopsis genes in parallel using high-density arrays of partially sequenced cDNA. The technology is simple and robust and reliably permits the detection of down to twofold variation in mRNA levels. The detection limit lies below 0.01% of the total mRNA population. The comparison of the profiles obtained for light-grown and dark-grown seedlings revealed significant variations in mRNA levels for about 16% of the cDNA investigated. This technology not only provides new functional information on anonymous genes, and thus may guide reverse-genetics approaches, but also constitutes a powerful tool for global gene expression studies, with many potential applications in plant biology.

Arabidopsis↗
PubMed1986

Deletion mutants that affect expression of Epstein-Barr virus nuclear antigen in COS-1 cells after gene transfer with simian virus 40 vectors containing portions of the BamHI K fragment.

We have identified sequences that affect the efficient expression of Epstein-Barr virus nuclear antigen (EBNA 1) when the structural portion of its gene, found within the 2.9-kilobase-pair BamHI/HindIII fragment called Ilf, is expressed from a simian virus 40 vector. A set of nested deletions at the BamHI end of the fragment was constructed by using BAL 31 digestion, the addition of linkers, and ligation into pSVOd. The mutants were tested for their ability to express antigen in COS-1 monkey cells by using indirect immunofluorescence and immunoblotting. Deletion endpoints were determined by DNA sequencing of the 5' ends of the mutants. The deletion mutants could be subclassified into four groups based on their ability to express EBNA polypeptide. Mutants that retain more than 106 base pairs upstream from the start of the open reading frame in Ilf exhibit antigen expression indistinguishable from that of wild type. Mutants that invade the structural gene by 1,115 or more bases destroy antigen expression. Mutants that alter the splice acceptor site or invade the open reading frame by a short distance make antigen at a markedly lower frequency. There are three mutants, whose deletions map at -78, -70, and -44 base pairs upstream of the open reading frame, that make reduced levels of EBNA. Since these three mutants differ in the extent to which EBNA expression is impaired, the data suggest that there are several critical regions upstream of the open reading frame that regulate EBNA expression in COS-1 cells. It is not known whether these regulatory sequences, which would be located in an intron in the intact genome, play any role in the expression of EBNA in infected lymphocytes.

Animals↗
PubMed1996

Adenovirus vector technology: an efficient method for constructing recombinant adenovirus and on/off switching of gene expression.

An efficient method of constructing recombinant adenoviruses (Ad) has been established. The expression unit to be introduced into recombinant Ad was first inserted into the unique SwaI site of the full-length Ad genome cloned in a cassette cosmid. The cassette bearing the expression unit was then cotransfected to 293 cells together with the Ad DNA-terminal protein complex digested at several sites with EcoT22I. The use of the parent Ad DNA-terminal protein complex instead of the deproteinized Ad genome DNA allowed very efficient recovery of the desired recombinant Ad, and the restriction digestion drastically reduced regeneration of the parent virus. This method may facilitate the application of recombinant Ad and should be useful for further improvement of Ad vectors. Also a recombinant adenovirus expressing Cre recombinase derived from bacteriophage P1 was constructed. To assay the Cre activity in mammalian cells, another recombinant Ad bearing an on/off switching reporter unit, where a LacZ-expression unit can be activated by the Cre-mediated excisional deletion of an interposed stuffer DNA, was also constructed. Co-infection experiments together with the Cre-expressing and the reporter recombinant Ad showed that the Cre-mediated switching of gene expression was detected in nearly 100% of cultured CV1 cells. These results suggest that the recombinant Ad efficiently expressed functional Cre and offers a basis for establishing a powerful on/off switching strategy of gene expression in cultured mammalian cells and presumably in future gene therapy.

Adenoviridae↗
PubMed2000

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats.

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.

Adenoviridae↗
PubMed1991

Transgenesis in fish.

Gene transfer into fish embryo is being performed in several species (trout, salmon, carps, tilapia, medaka, goldfish, zebrafish, loach, catfish, etc.). In most cases, pronuclei are not visible and microinjection must be done into the cytoplasm of early embryos. Several million copies of the gene are generally injected. In medaka, transgenesis was attempted by injection of the foreign gene into the nucleus of oocyte. Several reports indicate that the injected DNA was rapidly replicated in the early phase of embryo development, regardless of the origin and the sequence of the foreign DNA. The survival of the injected embryos was reasonably good and a large number reached maturity. The proportion of transgenic animals ranged from 1 to 50% or more, according to species and to experimentators. The reasons for this discrepancy have not been elucidated. In all species, the transgenic animals were mosaic. The copy number of the foreign DNA was different in the various tissues of an animal and a proportion lower than 50% of F1 offsprings received the gene from their parents. This suggests that the foreign DNA was integrated into the fish genome at the two cells stage or later. An examination of the integrated DNA in different cell types of an animal revealed that integration occurred mainly during early development. The transgene was found essentially unrearranged in the fish genome of the founders and offsprings. The transgenes were therefore stably transmitted to progeny in a Mendelian fashion. Southern blot analysis revealed the presence of possible junction fragments and also of minor bands which may result from a rearrangement of the injected DNA. In all species, the integrated DNA appeared mainly as random end-to-end concatemers. In adult trout blood cells, a small proportion of the foreign DNA was maintained in the form of non-integrated concatemers, as judged by the existence of end fragments. The transgenes were generally only poorly expressed. The majority of the injected gene constructs contained essentially mammalian or higher vertebrates sequences. The comparison of the expression efficiency of these constructs in transfected fish and mammalian cells indicates that some of the mammalian DNA sequences are most efficiently understood by the fish cell machinery. Chloramphenicol acetyl transferase gene under the control of promoters from Rous sarcoma virus, and human cytomegalovirus, was expressed in several tissues of transgenic fish. Chicken delta-crystallin gene was expressed in several tissues of transgenic fish.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2006

Overexpression of the genes from thermophiles in Escherichia coli by high-temperature cultivation.

Twenty-nine aminotransferase genes from Pyrococcus horikoshii, Aeropyrum pernix, and Sulfolobus tokodaii were cloned and expressed in Escherichia coli. The expression of several of the genes at 15, 25, or 37 degrees C resulted in the formation of insoluble protein aggregates. Therefore, we developed a simple method to express these genes into soluble proteins, by cultivating E. coli clones at a higher temperature. Thus, four genes could be expressed efficiently into soluble and active enzymes by cultivating the respective E. coli clones at 46 degrees C. Subsequently, the method was applied to the expression into soluble proteins of other aminotransferase genes that were derived from nine species of thermophilic microorganisms.

Aeropyrum↗
PubMed2005

The mouse vesicular inhibitory amino acid transporter gene: expression during embryogenesis, analysis of its core promoter in neural stem cells and a reconsideration of its alternate splicing.

The vesicular inhibitory amino acid transporter, VIAAT (also known as vesicular GABA transporter VGAT) transports GABA or glycine into synaptic vesicles. To initiate an analysis of the expression and regulation of VIAAT during neurogenesis we have cloned and characterized the mouse Viaat gene. We find that the mouse Viaat coding sequence is encoded by two exons spanning 5.3 kb. A survey of expression by whole mount in situ hybridization of mouse embryos indicates that Viaat is activated early in neuron differentiation and is expressed widely within the developing CNS; however, we did not detect expression in the superficial non-neural structures that express the GABA synthase Gad1. Analysis of the Viaat promoter indicates that a minimal promoter region containing a CG rich sequence is sufficient for efficient expression in neural stem and precursor cells. Our analysis of the Viaat sequence and splicing does not support the existence of two Viaat isoforms as previously proposed [Ebihara et al., Brain Res. Mol Brain Res. 110 (2003), 126-139]. Instead, the alternative isoform Viaat-a appears to be due to PCR artifacts that have occurred independently in multiple labs.

Alternative Splicing↗
PubMed2006

Stable inhibition of hepatitis B virus expression and replication in HepG2.2.15 cells by RNA interference based on retrovirus delivery.

RNA interference (RNAi) of virus-specific genes has emerged as a potential antiviral strategy. In order to suppress hepatitis B virus (HBV) expression and replication, a retrovirus-based RNAi system was developed, which utilized the U6-RNA polymerase III (Pol III) promoter to drive efficient expression and deliver the HBV-specific short hairpin RNAs (shRNAs) in HepG2.2.15 (2215) cells. In this system, the retrovirus vector with a puromycin selection marker was integrated into the host cell genome and allowed stable expression of shRNAs. In Puro-resistant 2215 cells, the levels of both HBV protein and mRNA were dramatically reduced by over 88% and HBV replication was suppressed. The results demonstrated that retrovirus-based RNAi technology will have foreseeable applications both in experimental biology and molecular medicine.

Gene Expression Regulation, Viral↗
PubMed2005

Expression and characterization of human vascular endothelial growth factor (VEGF165) in insect cells.

Vascular endothelial growth factor (VEGF) is the best characterized multifunctional protein which plays a key role in normal and pathologic angiogenesis. The gene encoding the human VEGF165 was cloned from the ovarian carcinoma cell line (OVCAR3) and expressed in insect cells using the baculovirus expression vector system. The recombinant human VEGF165 (rhVEGF165) protein produced by Sf21 (Spodoptera frugiperda) cells underwent a similar processing compared with mammalian cells, including efficient glycosylation, formation of a disulfide-linked dimer and secretion into the media. The rhVEGF165 had a high affinity for heparin and this characteristic was used to purify this form to homogeneity by heparin affinity, Resource S and Resource RPC columns. The biological activity of the purified 42-kDa homodimer was shown by the induction of the proliferation of human umbilical vein derived endothelial cells. These results demonstrate that an angiogenic growth factor whose normal processing requires glycosylation and disulfide-bridge formation can be efficiently expressed in high concentration (up to 20mg/L) in Sf21 cells.

Animals↗
PubMed2005

Vpu-mediated CD4 down-regulation and degradation is conserved among highly divergent SIV(cpz) strains.

Human immunodeficiency virus type 1 (HIV-1) along with simian immunodeficiency viruses from chimpanzees (SIV(cpz)) and three species of Old World monkeys from the genus Cercopithecus have been shown to encode a Vpu protein. To date, the functional characterization of Vpu has been limited to a small number of subtype B and more recently subtype C Vpu proteins. Using a recently developed VpuEGFP reporter system, we have shown that the subtype B and C Vpus are capable of preventing CD4 from being expressed on the cell surface. Using the same reporter system, we report here on the expression and functional analysis of Vpu protein from four SIV(cpz) isolates (CAM13, ANT, TAN1, and GAB1). All four SIV Vpu fusion proteins were efficiently expressed and prevented CD4 expression on the cell surface and induced CD4 degradation. This was surprising as three of the SIV(cpz) Vpu fusion proteins had only one canonical casein kinase II (CK-II) site (CAM13, ANT, TAN1) while previous studies with laboratory adapted HXB2 had indicated that both CK-II sites were required for CD4 degradation. Both ANT and TAN1 Vpu sequences encoded five consecutive negatively charged amino acids residues following the only CKII site (SAIEEDEE for ANT; SGVEEDEE for TAN1). We thus explored the possibility that this stretch of negatively charged amino acids might substitute for the lack of second CK-II site. Substitution of the aspartic acid at position 61 and glutamic acid at position 63 in the SIV(cpz) ANT Vpu within with lysine residues abolished the ability of this protein to down-modulate cell surface expression of CD4. Similarly, change of a serine to an alanine residue following the single consensus CK-II site of the CAM13 Vpu (SGNESDGGEEE) abolished CD4-down-regulation, suggesting that this serine was phosphorylated in the absence of a canonical CK-II site. Our results indicate that the serine was required, suggesting that this serine was phosphorylated by CK-II or possibly another cellular kinase. Taken together, these results show for the first time that Vpu proteins from SIV(cpz) isolates, although quite diverse in sequence and predicted secondary structure from the HIV-1 subtype B protein, are capable of down-regulating CD4, which is one of the major functions of the HIV-1 protein.

Amino Acid Sequence↗
PubMed2002

High-level expression of the C-terminal hydrophobic region of HCV E2 protein ectodomain in E. coli.

High-level expression of hepatitis C virus (HCV) E2 protein fragments encompassing its C-terminal hydrophobic region (downstream of aa 661) in Escherichia coli has been proven difficult. The extreme hydrophobicity of this region has been suspected to be detrimental to the host. In this work, we found that the C-terminal region (downstream of aa 565) of E2 ectodomain interfered with full-length expression of E2 fragments in E. coli. Nonetheless, when the central region (aa 484-622) of E2 was deleted, full-length protein was efficiently produced. C-terminal region aa 567-700 could not be efficiently expressed individually or as mouse dihydrofolate reductase (DHFR) fusion protein. However, a mutant that emerged in the cloning process was able to express full-length DHFR fusion protein. Sequencing analysis reveals the mutation to be a short frame-shift around the fusion junction, altering aa 568-571 of E2. C-terminal region of E2 ectodomain (aa 567-730) carrying this mutation was successfully expressed as hexa-histidine-tagged protein to a high level. The protein was highly insoluble and was purified under denaturing conditions. The purified protein displayed HCV E2-specific antigenicity in Western blot and specific rabbit antiserum was raised against it. These results demonstrate that hydrophobicity of the C-terminal region of E2 ectodomain is not harmful to E. coli host and has no dominative adverse effect on its bacterial expression. Other nucleotide and/or amino acid sequence properties seem to play a more important role. This finding opens up new possibilities for the development of novel bacterially-derived E2 proteins for research and clinical applications.

Animals↗
PubMed2006

An intron with a constitutive transport element is retained in a Tap messenger RNA.

Alternative splicing is a key factor contributing to genetic diversity and evolution. Intron retention, one form of alternative splicing, is common in plants but rare in higher eukaryotes, because messenger RNAs with retained introns are subject to cellular restriction at the level of cytoplasmic export and expression. Often, retention of internal introns restricts the export of these mRNAs and makes them the targets for degradation by the cellular nonsense-mediated decay machinery if they contain premature stop codons. In fact, many of the database entries for complementary DNAs with retained introns represent them as artefacts that would not affect the proteome. Retroviruses are important model systems in studies of regulation of RNAs with retained introns, because their genomic and mRNAs contain one or more unspliced introns. For example, Mason-Pfizer monkey virus overcomes cellular restrictions by using a cis-acting RNA element known as the constitutive transport element (CTE). The CTE interacts directly with the Tap protein (also known as nuclear RNA export factor 1, encoded by NXF1), which is thought to be a principal export receptor for cellular mRNA, leading to the hypothesis that cellular mRNAs with retained introns use cellular CTE equivalents to overcome restrictions to their expression. Here we show that the Tap gene contains a functional CTE in its alternatively spliced intron 10. Tap mRNA containing this intron is exported to the cytoplasm and is present in polyribosomes. A small Tap protein is encoded by this mRNA and can be detected in human and monkey cells. Our results indicate that Tap regulates expression of its own intron-containing RNA through a CTE-mediated mechanism. Thus, CTEs are likely to be important elements that facilitate efficient expression of mammalian mRNAs with retained introns.

Alternative Splicing↗
PubMed1996

Cell surface expression of the epithelial Na channel and a mutant causing Liddle syndrome: a quantitative approach.

The epithelial amiloride-sensitive sodium channel (ENaC) controls transepithelial Na+ movement in Na(+)-transporting epithelia and is associated with Liddle syndrome, an autosomal dominant form of salt-sensitive hypertension. Detailed analysis of ENaC channel properties and the functional consequences of mutations causing Liddle syndrome has been, so far, limited by lack of a method allowing specific and quantitative detection of cell-surface-expressed ENaC. We have developed a quantitative assay based on the binding of 125I-labeled M2 anti-FLAG monoclonal antibody (M2Ab*) directed against a FLAG reporter epitope introduced in the extracellular loop of each of the alpha, beta, and gamma ENaC subunits. Insertion of the FLAG epitope into ENaC sequences did not change its functional and pharmacological properties. The binding specificity and affinity (Kd = 3 nM) allowed us to correlate in individual Xenopus oocytes the macroscopic amiloride-sensitive sodium current (INa) with the number of ENaC wild-type and mutant subunits expressed at the cell surface. These experiments demonstrate that: (i) only heteromultimeric channels made of alpha, beta, and gamma ENaC subunits are maximally and efficiently expressed at the cell surface; (ii) the overall ENaC open probability is one order of magnitude lower than previously observed in single-channel recordings; (iii) the mutation causing Liddle syndrome (beta R564stop) enhances channel activity by two mechanisms, i.e., by increasing ENaC cell surface expression and by changing channel open probability. This quantitative approach provides new insights on the molecular mechanisms underlying one form of salt-sensitive hypertension.

Amino Acid Sequence↗
PubMed2005

Extracellular domain nicotinic acetylcholine receptors formed by alpha4 and beta2 subunits.

Models of the extracellular ligand-binding domain of nicotinic acetylcholine receptors (nAChRs), which are pentameric integral membrane proteins, are attractive for structural studies because they potentially are water-soluble and better candidates for x-ray crystallography and because their smaller size is more amenable for NMR spectroscopy. The complete N-terminal extracellular domain is a promising foundation for such models, based on previous studies of alpha7 and muscle-type subunits. Specific design requirements leading to high structural fidelity between extracellular domain nAChRs and full-length nAChRs, however, are not well understood. To study these requirements in heteromeric nAChRs, the extracellular domains of alpha4 and beta2 subunits with or without the first transmembrane domain (M1) were expressed in Xenopus oocytes and compared with alpha4beta2 nAChRs based on ligand binding and subunit assembly properties. Ligand affinities of detergent-solubilized, extracellular domain alpha4beta2 nAChRs formed from subunits with M1 were nearly identical to affinities of alpha4beta2 nAChRs when measured with [3H]epibatidine, cytisine, nicotine, and acetylcholine. Velocity sedimentation suggested that these extracellular domain nAChRs predominantly formed pentamers. The yield of these extracellular domain nAChRs was about half the yield of alpha4beta2 nAChRs. In contrast, [3H]epibatidine binding was not detected from the extracellular domain alpha4 and beta2 subunits without M1, implying no detectable expression of extracellular domain nAChRs from these subunits. These results suggest that M1 domains on both alpha4 and beta2 play an important role for efficient expression of extracellular domain alpha4beta2 nAChRs that are high fidelity structural models of full-length alpha4beta2 nAChRs.

Animals↗
PubMed1996

Particle-mediated gene transfer of granulocyte-macrophage colony-stimulating factor cDNA to tumor cells: implications for a clinically relevant tumor vaccine.

The necessity for prolonged tissue culture manipulations limits the clinical application of many form of gene therapy in patients with malignancies. We hypothesized that granulocyte-macrophage colony-stimulating factor (GM-CSF) cDNA in a plasmid expression vector could be effectively introduced into resting tumor cells, without the need for tissue culture propagation prior to or following transfection, and that efficient expression of transgenic GM-CSF by the transfected tumor cells would confer an effective immune response against tumors. GM-CSF cDNA in expression vectors was coated onto gold particles and accelerated with a gene gun device into mouse and human tumor cells. Human tumor tissue transfected within 4 hr of surgery produced significant levels of transgenic human GM-CSF protein in vitro. Human GM-CSF was readily detectable in serum and at the injection site following subcutaneous implantation of these transfected tumor cells into nude mice. Transfected and irradiated murine B16 melanoma cells produced > or = 100 ng/ml murine GM-CSF/10(6) cells per 24 hr in vitro for at least 10 days. The antitumor efficacy of this nonviral approach was tested using irradiated B16 tumor cells that were transfected with mGM-CSF cDNA and injected into mice as tumor "vaccine". Subsequent challenge of these mice with nonirradiated, nontransfected B16 tumor cells showed that 58% of the animals wer protected from the tumor by the prior vaccine treatment. In contrast, only 2% of control animals were protected by prior treatment with irradiated B16 cells transfected with the vector containing the luciferase gene. These results suggest that particle-mediated transfection of fresh tumor explants with cytokine cDNA is an effective and clinically attractive approach for cancer therapy.

Animals↗
PubMed1999

High-level expression of human lactoferrin in milk of transgenic mice using genomic lactoferrin sequence.

In our previous study, transgenic mice were generated that expressed human lactoferrin (hLF) in milk using cDNA under control of the 2 kb bovine beta-casein promoter. The expression level of the protein in milk of 7 mice ranged from 1 to 200 microg/ml; 1 to 34 microg/ml in 6 mice and 200 microg/ml in 1 mouse. With the aim of inducing higher expression of the protein, we constructed an expression cassette comprised of 10 kb of the bovine beta-casein gene promoter and the hLF genomic sequence in place of the cDNA. The hLF genomic sequence of about 27 kb, spanning 23 kb of the entire coding region and 4 kb of the 3'-flanking sequence, was placed downstream the bovine beta-casein promoter. In total, 8 transgenic mice were generated from 31 mice (transgenic rate of 25.8%) born from the embryos microinjected with the 40-kb hLF expression cassette. Mammary-specific expression of the transgene was addressed by performing Northern hybridization of the total RNAs from various tissues of transgenic mice. Immunoblot analysis showed that the recombinant protein expressed in milk has the same molecular weight as the native protein. The amount of the protein in milk of 5 mice ranged from 60 to 6,600 microg/ml when judged by ELISA analysis. Three mice expressed the protein at the level higher than 500 microg/ml. These data suggest that the genomic lactoferrin sequence represents a valuable element for the efficient expression of the protein in milk of transgenic animals.

Animals↗
PubMed1991

Expression of human immunodeficiency virus type 1 (HIV-1) gag, pol, and env proteins from chimeric HIV-1-poliovirus minireplicons.

Recent studies have demonstrated that genomes of poliovirus with deletions in the P1 (capsid) region contain the necessary viral information for RNA replication. To test the effects of the substitution of foreign genes on RNA replication and protein expression, chimeric human immunodeficiency virus type 1 (HIV-1)-poliovirus genomes were constructed in which regions of the gag, pol, or env gene of HIV-1 were substituted for regions of the P1 gene in the infectious cDNA clone of type 1 Mahoney poliovirus. The HIV-1 genes were inserted between nucleotides 1174 and 2956 of the poliovirus cDNA so that the translational reading frame was maintained between the HIV-1 genes and the remaining poliovirus genes. The chimeric genomes were positioned downstream from a T7 RNA polymerase promoter and transcribed in vitro by using T7 RNA polymerase, and the RNA was transfected into HeLa cells. A Northern (RNA blot) analysis of the RNA from transfected cells demonstrated the appropriate-size RNA, corresponding to the full-length chimeric genomes, which increased over time. Immunoprecipitation with antibodies specific for poliovirus RNA polymerase or sera from AIDS patients demonstrated the expression of the poliovirus RNA polymerase and HIV-1 proteins as fusions with the poliovirus P1 protein. The expression of the HIV-1-poliovirus P1 fusion protein was dependent upon an intact RNA polymerase gene, indicating that RNA replication was required for efficient expression. A pulse-chase analysis of the protein expression from the chimeric genomes demonstrated the initial rapid proteolytic processing of the polyprotein from the chimeric genomes to give HIV-1-poliovirus P1 fusion protein in transfected cells; the HIV-1 gag-P1 and HIV-1 pol-P1 fusion proteins exhibited a greater intracellular stability than the HIV-1 env-P1 fusion protein. Finally, superinfection with wild-type poliovirus of HeLa cells which had been transfected with the chimeric genomes did not significantly affect the expression of chimeric fusion protein. The results are discussed in the context of poliovirus RNA replication and demonstrate the feasibility of using poliovirus genomes (minireplicons) as novel vectors for expression of foreign proteins.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Genetic evidence that acute morphologic transformation, induction of cellular DNA synthesis, and focus formation are mediated by a single activity of the bovine papillomavirus E5 protein.

The bovine papillomavirus (BPV) type 1 E5 gene encodes a 44-amino-acid protein that can stably transform cultured rodent cells when expressed in the absence of all other viral genes. We have previously constructed a BPV-simian virus 40 recombinant virus (Pava-1) which efficiently expresses the BPV type 1 E5 gene in infected cells (J. Settleman and D. DiMaio, Proc. Natl. Acad. Sci. USA 85:9007-9011, 1988). Within 48 h of Pava-1 infection, the vast majority of mouse C127 cells underwent a dramatic morphologic transformation which was accompanied by cell proliferation. Infection of C127 cells made quiescent by contact inhibition and serum starvation caused a great induction of cellular DNA synthesis. These morphologic and mitogenic responses were proportional to the virus multiplicity of infection. Mutational analysis indicated that the E5 gene is both necessary and sufficient for these activities. Analysis of a variety of E5 missense mutants revealed a strong correlation between their phenotypes in the acute transformation assays following infection and in the stable focus-forming assay following transfection. Most of the defective mutants expressed normal levels of E5 protein following infection, indicating that their defective phenotypes are not due to the synthesis of an unstable protein. The failure to genetically resolve these E5 activities suggests that the ability of the E5 protein to cause acute morphologic transformation and reentry into the cell cycle may be intimately related to its ability to cause stable cell transformation and that these functions are probably mediated by a single biochemical activity of the E5 protein.

Amino Acid Sequence↗