Expression of a bacterial gene in plants mediated by infectious geminivirus DNA

A viable coat protein deletion mutant of cassava latent virus (CLV) DNA 1 has been isolated, suggesting that this geminivirus might be exploited as a gene replacement vector. An extensive deletion of 727 nucleotides within the coat protein gene renders DNA 1 non-infectious. Chimeric clones have been constructed in which the deleted coat protein open reading frame has been replaced by the coding region of the bacterial chloramphenicol acetyl transferase (CAT) gene. Infectivity is restored to DNA 1 when the CAT gene is inserted in either orientation, producing symptoms typical of CLV infection. The results demonstrate that the coat protein plays no essential role in virus spread throughout the host. Levels of CAT expression of 80 U/mg soluble protein occur in systemically infected Nicotiana benthamiana leaves when the CAT gene is fused in-frame to the amino terminus of the coat protein, providing a sensitive assay for viral DNA replication.     

The organization of the rat GRP78 gene and A23187-induced expression of fusion gene products targeted intracellularly

Expression of the glucose-regulated protein, GRP78, is markedly increased when cells are placed in a variety of stressful environments (i.e., low glucose medium, calcium ionophore treatment). In this report, the genomic organization of the rat GRP78 gene is described. This gene comprises eight exons and encodes a protein which is highly hydrophilic with the notable exception of several short hydrophobic domains. The first hydrophobic region, 18 amino acids at the N-terminus of the protein, putatively acts as a signal sequence to target GRP78 into the endoplasmic reticulum (ER). By ligating portions of the GRP78 gene and its promoter to the bacterial gene encoding chloramphenicol acetyltransferase (CAT), we created heterologous CAT genes inducible by calcium ionophore A23187. Through immunofluorescence analysis, the intracellular localizations of endogenous GRP78 and fusion CAT proteins under normal growth and A23187-induced conditions are identified. By fusing the GRP78 signal sequence to CAT, we influence intracellular targeting of the CAT protein into the ER.     

Tissue-specific expression in transgenic mice directed by the 5'-flanking sequences of the human gene encoding interphotoreceptor retinoid-binding protein

Interphotoreceptor retinoid-binding protein (IRBP) is an extracellular protein that has been suggested to participate in the visual process as a carrier for visual retinoids. A chimeric gene composed of the human IRBP promoter fused to the bacterial reporter gene chloramphenicol acetyltransferase (CAT) was used to generate transgenic mice. Analysis of six transgenic families revealed that the CAT gene, concomitant with the endogenous IRBP gene, was expressed primarily in the retina and, to a lesser extent, in the pineal gland. These results establish that a 1.3-kilobase fragment from the 5' end of the human IRBP gene is sufficient to direct transgene expression to a visual subdivision of the central nervous system.     

Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo.

We have constructed recombinant adenoviruses (Ad), with functional or defective E1a genes, which harbor either the hepatitis B (HB) virus s gene encoding the HB surface antigen, as well as the pre-S2 epitopes, or the bacterial gene encoding chloramphenicol acetyltransferase (CAT) under control of the Ad major late promoter (MLP). The recombinant viruses defective for E1a (Ad.MLP.S2 and Ad.CAT), which can be efficiently propagated only on 293 cells that complement this defect, and the nondefective (Ad.MLP.S2.E1A) recombinant were used to infect a wide spectrum of cells of different origin. The yields of HBs and CAT proteins obtained with these different recombinant viruses demonstrate no real advantage to using nondefective vectors, whatever the cell type infected. The injection into chimpanzees of Ad.MLP.S2 does not elicit the production of antibodies, but can immunologically prime the animals, resulting in a partial protection against HBV challenge.     

Expression of a foreign protein by influenza A virus.

In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT). The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein. The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences. The resultant gene produces a polyprotein which is proteolytically cleaved to release both CAT and NA. The intramolecular cleavage occurs at the C terminus of the 2A sequence between a glycine-proline dipeptide motif such that the released NA protein has an additional N-terminal proline residue. The transfectant virus is stable upon passage in tissue culture. CAT activity is expressed at high levels in cell culture supernatants and in the allantoic fluid of infected eggs. Since the chimeric segment must maintain the heterologous reading frame to retain viability, the virus stability is dependent upon concomitant synthesis of the heterologous protein. This design may be particularly appropriate for utilization of influenza virus as a mammalian expression vector.     

Expressing an antibacterial protein in bacteria for raising antibodies

Magainins are small peptides with broad-spectrum activity against a range of plant and animal microbial pathogens. To detect magainin peptides in applications such as Western blot analysis and enzyme-linked immunosorbent assays, specific antibodies that recognize magainin peptides are required. The production of antibodies against small peptides injected into host animals poses problems with respect to eliciting an adequate immunogenic response due to the small size of the molecules. To increase the immunogenicity of a target peptide, it may be expressed as part of a larger fusion protein. However, expression of an antimicrobial peptide in bacteria may be cytotoxic to the host or subjected to degradation by host-derived peptidases. To overcome these potential problems, we fused the DNA coding sequence of a magainin gene analogue within the sequence of a bacterial thioredoxin gene. The subsequent gene fusion comprising a bacterial thioredoxin gene with a magainin coding sequence ligated at the active site of thioredoxin was successfully translated in a bacterial expression system. The fusion protein was non-toxic to the host bacteria. This represents a novel strategy to express antimicrobial peptides in a bacterial expression system. The fusion protein, purified by molecular size separation, was recovered in a soluble form following electroelution from polyacrylamide gels. Sufficient fusion protein was obtained for injection into rabbits and antibodies were obtained from rabbit sera that selectively recognized magainin peptides in Western blot analysis.     

Receptor-mediated gene delivery and expression in vivo

A soluble DNA carrier system was used to target a foreign gene specifically to liver in vivo via asialoglycoprotein receptors. The DNA carrier was prepared consisting of a galactose-terminal (asialo-)glycoprotein, asialoorosomucoid (AsOR), covalently linked to poly-L-lysine. The conjugate was complexed in a 2:1 molar ratio (based on AsOR content of the conjugate) to the plasmid, pSV2 CAT, containing the gene for the bacterial enzyme chloramphenicol acetyltransferase (CAT). Intravenous injection of [32P]plasmid DNA complexed to the carrier demonstrated specific hepatic targeting with 85% of the injected counts taken up by the liver in 10 min compared to only 17% of the counts when the same amount of [32P]DNA alone was injected under identical conditions. Targeted pSV2 CAT DNA was detected at a level of 1.0 ng/g liver by hybridization of a [32P]pSV2 CAT cDNA probe to rat liver DNA extracted 24 h after intravenous injection of AsOR-poly-L-lysine-DNA complex containing 1.0 mg of DNA. Homogenates of livers taken 24 h after injection of the complex revealed that the targeted CAT gene was functional as reflected by the detection of CAT activity (approximately 4 microunits/mg protein). Livers from control animals that received individual constituents of the complex produced no CAT activity. Simultaneous injection of excess AsOR to compete with the AsOR-poly-L-lysine-DNA complex for uptake by the liver inhibited CAT gene expression. Assays for CAT activity in other organs (spleen, kidney, lungs) failed to demonstrate any activity in these organs. This new soluble DNA carrier system can permit targeted delivery of foreign genes specifically to liver with resultant foreign gene expression in vivo.     

Expression and characterization of the tau subunit of phosphorylase kinase

A cDNA encoding the entire tau subunit of rabbit skeletal muscle phosphorylase kinase was reconstructed and inserted into a plasmid containing the Escherichia coli ptac promoter and a constructed plasmid containing the ptac promoter and bacterial chloramphenicol acetyl transferase (CAT) gene, respectively. A significant phosphorylase kinase activity was found, in the first case. In the second case, a fused protein containing 73 amino acids from the CAT protein was obtained. After renaturation, the CAT-tau subunit protein shows enzymatic activity similar to the HPLC-purified and renatured tau subunit.     

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.     

Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene

The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.     

An adjustable expression system for controlling growth rate, plasmid maintenance, and culture dynamics

Recombinant bacterial cells express various levels of model product proteins if the genes of interest are regulated by controllable promoters. The level of gene expression influences the growth-rate differential between plasmid-bearing and plasmid-free cells, and thereby affects the culture dynamics of a plasmid-containing cell population. An expression system has been designed in which host Escherichia coli cells contain the pil operon controlled by a tac promoter; these cells are transformed with plasmids that contain the repressor gene, lacl, for the tac promoter, in combination with an expression system for a model protein, chloramphenicol acetyl tranferase (CAT). Experimental and theoretical results show that plasmid-bearing cells can be maintained as dominant in continuous cultures without selective pressure when 12% or less of the cells' total protein is the model product protein, CAT. This is because the segment cells produce pili greatly in excess of normal wild-type levels, and thus have more of a metabolic burden than do the plasmid-bearng cells that overproduce CAT. However, when the level of the plasmid-directed CAT expression is increased above 12% of the cells' total protein, the growth rate of the plasmid-bearing cells decreases to a value lower than that of the segregant cells. Therefore, plasmid-containing cells lose their selective advantage at this expression level, and cannot be maintained as the dominant cell type in a continuous culture unless antibiotic or other positive selection methods are used. By controlling the growth rate differential of this bacterial host/plasmid system, a variety of interesting competitive culture dynamics is investigated. All experimental measurements for continuous cultures are in very good agreement with theory using kinetic parameters determined from independent batch experiments. (c) 1992 John Wiley & Sons, Inc.     

Dual control of C-reactive protein gene expression by interleukin-1 and interleukin-6.

Human C-reactive protein (CRP) is the major acute phase reactant during acute inflammation. The human CRP promoter is expressed in an inducible and cell-specific manner when linked to the bacterial CAT gene and transfected into human hepatoma cell cultures. In this paper we analyze the effect of several recombinant cytokines or CRP promoter inducibility in human Hep3B cells. When cytokines are tested singly the major inducer of CRP-CAT fusions is interleukin-6 (IL-6). Maximal CAT gene expression, however, is only achieved when both interleukin-1 beta (IL-1 beta) and IL-6 are present. The response to the two cytokines is cooperative. Cooperativity is maintained when the CRP promoter is linked to a different coding region, that of the bacterial neomycin phosphotransferase II gene. With a series of 5' and 3' deletions we show the existence of two distinct and independent regions responsive to IL-6 and located upstream to the TATA box. The IL-1 effect is exerted at the level of downstream sequences that are probably important for optimal mRNA translatability or nuclear-cytoplasmic transport. Inducibility is not influenced by the activation of protein kinases C or A and does not require new protein synthesis.