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PubMed1992

Transient expression assay in a baculovirus system using firefly luciferase gene as a reporter.

Transient gene expression assays were developed to assess the function of the regulatory sequences of baculoviruses Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa californica nuclear polyhedrosis virus (AcNPV) in insect cells of Bombyx mori and Spodoptera frugiperda, respectively. DNA sequences encoding luciferase (luc) of the firefly Photinus pyralis was successfully employed in the expression assay as a reporter gene. Recombinant plasmids were constructed containing the luc gene under control of baculovirus-specific or heterologous promoters. Cotransfection of Bombyx mori and Spodoptera frugiperda cells with recombinant plasmids carrying virus-specific promoter sequences and BmNPV and AcNPV DNA, respectively, gave rise to efficient synthesis of luciferase (Luc), while heterologous promoters induced a low level of luc expression. We found that flanking sequences of the AcNPV DNA in the transfer plasmid contained an unknown promoter conferring an efficient luc expression. The activity of this promoter was modulated by the polh promoter sequences. The assay allows one to conduct highly sensitive monitoring of the transient expression of foreign genes from the transfecting plasmids prior to construction of recombinant viruses.

Animals↗
PubMed1990

Analysis of promoter activity from an alpha-zein gene 5' flanking sequence in transient expression assays.

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5' flanking region of a 19 kDa alpha-zein gene. For these analyses, the zein promoter region was fused to the beta-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5' of the zein promoters. Zein upstream sequences enhanced transcription independently of the -189/-114 region. Although the -189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5' of the zein promoter sequences. However, nucleotides -347 to -309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5' of deletion mutants of the zein promoter also failed to produce GUS activity above background.

Base Sequence↗
PubMed1991

Transient expression of neurotensin mRNA in the mitral cells of rat olfactory bulb during development.

The transient expression of neurotensin mRNA in the mitral cells of the rat olfactory bulb was demonstrated during the perinatal period using non-radioactive in situ hybridization in which an alkaline phosphatase labelled oligodeoxynucleotide probe was used. The relative cellular content of neurotensin mRNA signal was measured by use of a microdensitometer. Neurotensin mRNA positive cells were observed in the primordium of mitral cells on embryonic day 14 and their mRNA content increased gradually up to the day of birth. During the first postnatal week, the strength of their neurotensin mRNA signal decreased dramatically, and continued to decrease until in the adult olfactory bulb neurotensin mRNA was no longer detectable. This decrease of the neurotensin mRNA content coincided with a parallel decrease of neurotensin immunoreactivity observed in the lateral olfactory tract.

Animals↗
PubMed1990

Analysis of leaky viral translation termination codons in vivo by transient expression of improved beta-glucuronidase vectors.

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

Base Sequence↗
PubMed1987

A mechanism by which adenovirus virus-associated RNAI controls translation in a transient expression assay.

The mechanism by which adenovirus virus-associated RNAI stimulates translational efficiency in a transient-expression assay in 293 cells was investigated. We showed that DNA transfection leads to activation of a protein kinase that phosphorylates the alpha subunit of eucaryotic initiation factor 2 and, as a consequence, inhibition of polypeptide chain initiation. Cotransfection of a plasmid encoding adenovirus type 2 virus-associated RNAI recovered the translational capacity by preventing activation of the kinase.

Adenoviruses, Human↗
PubMed1989

Production of pro-opiomelanocortin (POMC) by a vaccinia virus transient expression system and in vitro processing of the expressed prohormone by POMC-converting enzyme.

Pro-opiomelanocortin (POMC) was expressed in CV-1 (green monkey kidney) cells using a vaccinia virus transient expression system [(1986) Proc. Natl. Acad. Sci. USA 83, 8122]. The system involved infection of cells with a recombinant vaccinia virus carrying the T7 RNA polymerase gene and transfection with a plasmid containing the mouse POMC sequence flanked by the T7 RNA polymerase promoter at its 5'-end and the T7 RNA polymerase terminator at its 3'-end. Assay of the medium from transfected cells showed that 1-2 micrograms of immunoreactive ACTH was produced/10(6) cells. Analysis of the same medium by SDS-PAGE/Western blots revealed a band of 30-36 kDa, which was immunostained with both ACTH and beta-endorphin antisera. Labeling the transfected cells with [3H]Arg, followed by immunoprecipitation and SDS-PAGE showed the synthesis of a major peak of POMC, 33 kDa. Purified [3H]POMC expressed by CV-1 cells was cleaved in vitro by bovine intermediate lobe secretory vesicle pro-opiomelanocortin-converting enzyme to ACTH intermediates (19-25 kDa), beta-lipotropin and beta-endorphin. Thus, this work has demonstrated a technique for expressing microgram quantities of prohormones in mammalian cells, suitable for use as substrates for prohormone-converting enzymes in vitro.

Adrenocorticotropic Hormone↗
PubMed1992

Association interneurons of embryonic rat spinal cord transiently express the cell surface glycoprotein SNAP/TAG-1.

SNAP/TAG-1 is a 135 kDa glycoprotein of the immunoglobulin superfamily that is transiently expressed upon the surfaces of developing axons. In the embryonic rodent spinal cord, this molecule is expressed by motor neurons, dorsal root ganglion cells, and commissural neurons (Yamamoto et al.: J. Neurosci. 6:3576-3594, 1986; Dodd et al.: Neuron 1:105-116, 1988). The commissural cells are a subset of early-forming dorsal horn interneurons whose axons follow a circumferential course in the embryonic spinal cord. The axons of commissural neurons cross the developing ventral commissure to terminate on contralateral synaptic targets, whereas those of the other subset of circumferential cells, the association interneurons, remain on the same side of the spinal cord to form ipsilateral, terminal synaptic fields. The difference between the axonal trajectories of these two subsets of nerve cells raised the question of whether or not association interneurons would also express the SNAP/TAG-1 epitope and, if so, how would this expression be related to that of the commissural cells. Immunocytochemistry for SNAP/TAG-1 and choline acetyltransferase (ChAT) was used to answer these questions. The results indicated that association interneurons expressed SNAP/TAG-1 epitopes and that this expression began later and lasted longer than that of the commissural neurons. Other new findings of this study included the identification of a lateral subgroup of commissural fibers that expressed SNAP/TAG-1 later than their more medially located counterparts, and these lateral fibers were more pronounced in the thoracic spinal cord than at cervical levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Transient expression of beta-glucuronidase in Arabidopsis thaliana leaves and roots and Brassica napus stems using a pneumatic particle gun.

Successful transient expression of beta-glucuronidase (GUS) in Arabidopsis thaliana leaves and roots and Brassica napus stems was obtained after gene delivery with a pneumatic particle gun driven by compressed air. Effects of the pneumatic pressure used to accelerate the particles (accelerating pressure; 85 to 200 kg/cm2) and of preculture periods of plant tissues (0 to 6 days) on the efficiency of gene delivery were studied. In A. thaliana leaves, best results were obtained at 115 kg/cm2 of accelerating pressure and 3 days of preculture. In A. thaliana roots, the optimum was at 200 kg/cm2 of accelerating pressure and 3 days of preculture. These results indicate that both preculture period and accelerating pressure are vital factors that determine the efficiency of gene delivery by particle gun.

Atmospheric Pressure↗
PubMed1989

Transient expression of a transfected gene in cultured epidermal keratinocytes: implications for future studies.

We examine the effect of keratinocyte differentiation upon transient expression of a nonepithelial gene following DNA-mediated transfer. Cultures of primary epidermal keratinocytes were transfected with the reporter gene, chloramphenicol acetyltransferase (CAT). The CAT gene was linked at the 5' end to the long terminal repeat (LTR) regulatory sequences from Rous sarcoma virus, and gene transfer was accomplished by the calcium phosphate coprecipitation method. Transfected cells were fractionated on Ficoll 400 density gradients. The major finding of this study was that the larger, more differentiated cells displayed five- to seven-fold higher levels of CAT activity per cell than the smaller, less differentiated cells. The higher levels of CAT activity did not result from greater uptake of DNA because cells of all gradient fractions contained one to two copies of plasmid DNA per cell. Furthermore, the CAT gene linked to the regulatory sequences from another virus, SV40, gave the same result. We conclude that the CAT gene, when controlled by these viral regulatory sequences, is expressed more efficiently in differentiated keratinocytes. These results have important implications for the interpretation of future studies of gene expression in transfected keratinocytes.

Avian Sarcoma Viruses↗
PubMed1989

Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb.

Structural proteins of the intermediate filament family are an early indicator of differentiation before organogenesis becomes apparent. Keratin intermediate filaments are characteristically expressed only by epithelial and not by mesenchymal cells. Here we show, using monoclonal antibodies, a transient expression of the keratin pair 8 and 18 in a population of mesenchymal cells in the regenerating newt limb, specifically in the undifferentiated progenitor cells (blastemal cells) which give rise to the new tissues. These keratins are also expressed in cultured limb cells that can differentiate into muscle. In contrast no reactivity with anti-keratin 8 and 18 antibodies was observed in the newt limb bud at an early stage of development, indicating a molecular difference between the developing and regenerating limb. The molecular weights of the newt proteins detected by these antibodies are very similar to those of human keratins 8 and 18, further supporting the immunocytochemical evidence that the newt homologs of these keratins are expressed in blastemal cells. This is the first demonstration of keratin expression in mesenchymal progenitor cells in an adult animal.

Animals↗
PubMed1992

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Animals↗
PubMed1990

Transient expression directed by homologous and heterologous promoter and enhancer sequences in fish cells.

In order to construct fish specific expression vectors for studies on gene regulation in vitro and in vivo a variety of heterologous enhancers and promoters from mammals and from viruses of higher vertebrate cells were tested for expression of the bacterial chloramphenicol acetyl transferase reporter gene in three teleost fish cell lines. Several viral enhancers were found to be constitutively active at high levels. The human metallothionein promoter showed inducible expression in the presence of heavy metal ions. A fish sequence was isolated that can be used as a homologous constitutively active promoter for expression of foreign genes. Using the human growth hormone gene with an active promoter in fish cells for transient expression insufficient splicing and lack of translation were observed, pointing to limitations in the use of heterologous genes in gene transfer experiments. On the contrary, some heterologous promoters and enhancers functioned in fish cells as well as in their cell type of origin, indicating that corresponding transcription factors are sufficiently conserved between fish and human over a period of 900 million years of independent evolution.

Animals↗
PubMed1992

Binding of typical and atypical antipsychotic agents to transiently expressed 5-HT1C receptors.

We determined the affinities of clozapine and 21 other typical and atypical antipsychotic agents for the cloned 5-hydroxytryptamine-1C (5-HT1C) receptor. For these studies, 5-HT1C receptors were transiently expressed in COS-7 cells using the vector pSVK3-5HT1C. We discovered that clozapine and several other putative typical and atypical antipsychotic agents (loxapine greater than tiosperone greater than SCH23390 greater than fluperlapine greater than rilapine greater than chlorpromazine) had relatively high affinities (7-30 nM) for the cloned 5-HT1C receptor. Other antipsychotic agents (risperidone greater than tenilapine greater than mesoridazine greater than thioridazine greater than cis-fluphenthixol) had intermediate affinities (30-100 nM), whereas many other antipsychotics (fluphenazine greater than spiperone greater than amperozide greater than melperone greater than thiothixene greater than haloperidol, metoclopramide, pimozide, domperidone, sulpiride) had low affinities (greater than 500 nM) for the cloned 5-HT1C receptor. The results indicate that although several putative atypical antipsychotic agents have high affinities for the cloned rat 5-HT1C receptor, the spectrum of drug binding does not correlate with the atypical nature of these compounds.

Antipsychotic Agents↗
PubMed1991

The transient expression of type II collagen at tissue interfaces during mammalian craniofacial development.

Using immunocytochemical techniques, the spatiotemporal distribution of the major collagen isoform of cartilage, type II collagen, has been investigated during early craniofacial development in the mouse embryo. Early and transient expression was associated with the otic and optic vesicles, the ventrolateral surfaces of the developing brain, olfactory conchi, endocardial and mesocardial tissues, the lateral and basal surfaces of the pharyngeal endoderm and beneath the ectoderm of the branchial arches. A number of these locations are sites of epithelial-mesenchymal tissue interaction believed to generate the component parts of the chondrocranium; here, type II collagen appears transiently in advance of overt chondrogenesis in the mesenchyme. At such sites, immunofluorescence is typically localised along the basal surface of the epithelial partner, with the strongest reaction detected between the basal aspects of the otic and rhombencephalic epithelia. Immunoelectron microscopy, using pre-embedding immunostaining and a protein G-gold technique, reveals that the type II collagen is adjacent to, but not integral with, the basal laminae. Gold particles are clearly associated with 10-15 nm fibrils of the extracellular matrix in the reticulate lamina region. The pattern of type II collagen expression in the mouse closely correlates with that demonstrated previously in the quail, indicating a high degree of phylogenetic conservation between these two vertebrate species. These findings are consistent with the hypothesis that the pattern of epithelial secretion of type II collagen, or a coexpressed matrix molecule, constitutes a morphogenetic signal, realised as a matrix-mediated tissue interaction, and specifying the form of the vertebrate chondrocranium. Three-dimensional reconstruction of early type II collagen distribution, and of the subsequent chondrocranial cartilages, reveals that chondrocranial form can be derived from a 'pre-pattern' of epithelially derived type II collagen expressed at epithelial-mesenchymal tissue interfaces.

Animals↗
PubMed1991

Transient expression analyses of DNA extending 2.4 kb upstream of the human renin gene.

The influence on homologous and heterologous promoter activity of DNA extending 2.4 kb upstream of the human renin gene (REN) was examined by transient expression assay in JEG-3 cells, using the gene for chloramphenicol acetyl transferase (CAT) as reporter, and cotransfection with pCH110 to control for transfection efficiency. Analyses of constituent subfragments of the region 5' of residue -144, using the herpes simplex virus thymidine kinase (tk) promoter to drive transcription, provided no evidence for negative regulatory influences within the -2400 to -144 DNA. That distal 5'-flanking DNA may have little influence on promoter activity is further supported by a sharp decline in nucleotide homology between human, rat and mouse renin genes further upstream than human residue -604. Constructs containing renin DNA to residue +13, i.e., which retained the REN promoter, all displayed very low CAT activities, consistent with negative cis-acting control within the -149 to +13 region. This finding contrasts with results of similar studies for mouse, in which renin gene control was suggested to be mediated primarily via cell-specific trans-acting activator(s) acting on yet-to-be identified enhancer(s). Mouse renin genes have, however, a common DNA insertion that could have disrupted the negative element in this region, and which might contain enhancer target(s) for trans-acting factor(s). In conclusion, the present study involving JEG-3 cells has demonstrated that distal human renin 5'-flanking DNA has little cis-acting influence on promoter activity, whereas DNA located within 100 base pairs of the renin promoter may have a negative regulatory effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning, Molecular↗
PubMed1992

Infectious Sindbis virus transient expression vectors for studying antigen processing and presentation.

Sindbis virus (SIN) is a small positive-strand enveloped RNA virus that infects a broad range of vertebrate and insect cells. A SIN vector (called dsSIN), designed for transient expression of heterologous RNAs and proteins, was engineered by inserting a second subgenomic mRNA promoter sequence into a nonessential region of the SIN genome. By using this vector, dsSIN recombinants have been constructed that express either bacterial chloramphenicol acetyltransferase, a truncated form of the influenza hemagglutinin (HA), or mini-genes encoding two distinct immunodominant cytotoxic T lymphocyte (CTL) HA epitopes. Infection of murine cell lines with these recombinants resulted in the expression of approximately 10(6)-10(7) chloramphenicol acetyltransferase polypeptides per cell and efficient sensitization of target cells for lysis by appropriate major histocompatibility complex-restricted HA-specific CTL clones in vitro. In addition, priming of an influenza-specific T-cell response was observed after immunizing mice with dsSIN recombinants expressing either a truncated form of HA or the immunodominant influenza CTL epitopes. This SIN expression system allows the generation of high-titered recombinant virus stocks in a matter of days and should facilitate mapping and mutational analysis of class I major histocompatibility complex-restricted T-cell epitopes expressed via the endogenous pathway of antigen processing and presentation.

Amino Acid Sequence↗
PubMed1990

Transient expression of full-length and truncated forms of the human nerve growth factor receptor.

To facilitate the characterization of nerve growth factor (NGF) receptor and mutated forms of the receptor, we have set up a rapid, efficient transient expression system utilizing COS cells. The human NGF receptor is a 427 amino acid protein with a hydrophobic signal sequence, a 222 amino acid extracellular domain, a single transmembrane domain and a 155 amino acid intracellular domain. The NGF receptor and a truncated form lacking the cytoplasmic domain were expressed in a COS cell expression system. Both recombinant proteins were detected on the cell surface and at a perinuclear site. Specific binding of 125I-NGF to the recombinant proteins was detected by chemical cross-linking. The extracellular domain of the NGF receptor was also expressed in the same system and detected in the COS cell endoplasmic reticulum and in the culture supernatant. This recombinant protein also specifically binds NGF.

Animals↗