Coli Chloramphenicol Acetyltransferase Research Articles

Efficient and scalable method for scaling up cell free protein synthesis in batch mode

A novel method for general cell free system scale-up in batch mode was applied to expression of E. coli chloramphenicol acetyl transferase (CAT) and a GMCSF-scFv fusion protein being developed as a B-cell lymphoma vaccine candidate (GLH). Performance of two different E. coli based cell-free systems was evaluated using the new scale-up approach. Reaction volumes from 15 to 500 microL were tested for both products and both reaction systems. In each case, the new scale-up method preserved total, soluble, and active volumetric yields of GLH and CAT at every reaction volume. At the 500 microL reaction volume, the PANOx SP system produced 560 +/- 36 microg/mL of active CAT and 99 +/- 10 microg/mL of active GLH protein using the new thin film approach whereas 500 microL test tube reactions produced 250 +/- 42 microg/mL and 72 +/- 7 microg/mL of active CAT and GLH respectively. Similarly, 500 microL cell-free synthesis reactions with the Cytomim system produced 481 +/- 38 microg/mL of active CAT and 109 +/- 15 microg/mL active GLH respectively in thin films compared to 29 +/- 7 microg/mL of active CAT and 5 +/- 2 microg/mL of active GLH protein in 500 microL test tube reactions. The new thin film approach improves oxygen supply for the Cytomim system, and increases the availability of hydrophobic surfaces. Analysis suggests that these surfaces provide significant benefit for protein expression and folding. We believe that this approach provides a general reaction scale-up technology that will be suitable for any protein target, cell free system, and reaction volume.

504. Gene Transfection into Fetal Sheep Airway Epithelium Using Cationic BGTC/DOPE Liposomes

Over the last years, we have developed a particular class of nonviral vectors, cationic cholesterol derivatives characterized by polar headgroups with guanidinium functions. Indeed, guanidinium groups have highly favorable features for DNA binding. Liposome formulations of bis(guanidinium)-tren-cholesterol (BGTC) with dioleoylphosphatidylethanolamine (DOPE) were found to be efficient for gene transfection into primary human airway epithelial cells as well as into the mouse airway epithelium in vivo by intratracheal administration (Proc. Natl. Acad. Sci. USA 1997, 94, 1651-1656). We have also shown that colloidal stabilization (via lipophilic polyethyleneglycol derivatives) of concentrated solutions of BGTC/DOPE lipoplexes allowed enhanced transfection of the mouse airways by intranasal instillation (J. Gene Med. 2001, 3, 478-487). Thus, as prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we recently undertook to explore the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero. Practically, pCIK-CAT plasmids expressing the E. coli chloramphenicol acetyltransferase (CAT) reporter gene (kindly provided by D. Gill, Oxford, UK) were complexed with BGTC/DOPE liposomes and the resulting lipoplexes were administered to sheep fetuses at 70 days of gestation via surgical replacement of the fetal airway fluid by the transfection mixture followed by tracheal ligature. The fetal tracheas and lungs were harvested at 72 hours and examined for CAT expression and evidence of toxicity. Using an ELISA assay, we detected significant CAT levels in lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked pCIK-CAT plasmid. Immunohistochemistry analysis showed that airway epithelial cells (at both the tracheal and bronchial levels) as well as some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed and manifested as focal epithelial and mesenchymal lesions. These results demonstrated that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium in utero and they also invited the development of optimized BGTC-based formulations with a view to future experiments with therapeutic genes. Thus, we are at present investigating the utility of BGTC-based lipoplexes for transferring genes encoding pulmonary growth factors in the fetal lamb model of congenital diaphragmatic hernia (CDH). In these ongoing experiments, a first surgical step allows the creation of the diaphragmatic hernia, whereas the second step consists of the substitution of the fetal airway fluid by the lipoplex medium followed by tracheal occlusion via a balloon. Nonviral gene delivery approaches may be particularly attractive here as long-term transgene expression is probably not required for the treatment of CDH before birth.

Gene transfection into fetal sheep airways in utero using guanidinium-cholesterol cationic lipids.

Over the last several years, we have developed a novel class of cationic lipids, cholesterol derivatives characterized by polar head groups with guanidinium functions. We have in particular shown that bis(guanidinium)-tren-cholesterol/dioleoylphosphatidylethanolamine (BGTC/DOPE) cationic liposomes can mediate efficient gene transfection into the mouse airways in vivo via direct intratracheal administration or intranasal instillation. As prenatal gene therapy may be necessary for the treatment of a variety of congenital lung diseases, we have explored in the present work the feasibility of BGTC-mediated gene transfection into the respiratory tract of fetal sheep in utero. Thus, BGTC/DOPE liposomes were complexed with plasmids expressing the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene and the resulting lipoplexes were administered to fetal sheep at 70 days of gestation via surgical replacement of the airway fluid by the transfection mixture followed by tracheal occlusion. The fetal lungs and tracheas were harvested at 72 h and examined for CAT expression and evidence of toxicity. CAT expression was detected in both lung and trachea homogenates, no CAT expression being observed in control fetuses receiving naked plasmid DNA. Immunohistochemical analysis showed that airway epithelial cells and some mesenchymal cells were transfected. Pulmonary histopathology of varied severity was however observed under our transfection conditions and manifested as focal epithelial and mesenchymal lesions. These results show that BGTC/DOPE liposomes can mediate gene transfection into the fetal sheep airway epithelium. They also invite the development of optimized BGTC-based formulations and administration conditions with a view to future prenatal gene transfer experiments involving therapeutic genes.

Anomalous RNA substrates for mammalian tRNA 3′ processing endoribonuclease

Mammalian tRNA 3′ processing endoribonuclease (3′ tRNase) is an enzyme responsible for the removal of a 3′ trailer from pre-tRNA. The enzyme can also recognize and cleave any target RNA that forms a pre-tRNA-like complex with another RNA. To investigate the interaction between 3′ tRNase and substrates, we tested various anomalous pre-tRNA-like complexes for cleavage by pig 3′ tRNase. We examined how base mismatches in the acceptor stem affect 3′ tRNase cleavage of RNA complexes, and found that even one base mismatch in the acceptor stem drastically reduces the cleavage efficiency. Mammalian 3′ tRNase was able to recognize complexes between target RNAs and 5′-half tDNAs, and cleave the target RNAs, although inefficiently, whereas the enzyme had no activity to cleave phosphodiester bonds of DNA. A relatively long RNA target, the Escherichia coli chloramphenicol acetyltransferase (CAT) mRNA, was cleaved by 3′ tRNase in the presence of appropriate 5′-half tRNAs. We also demonstrated that an RNA complex of lin-4 and lin-14 from Caenorhabditis elegans can be recognized and cleaved by pig 3′ tRNase.

Cytotoxic T lymphocyte responses to proteins encoded by heterologous transgenes transferred in vivo by adenoviral vectors.

Although replication-deficient adenovirus (Ad) vectors are efficient vehicles for in vivo gene transfer, persistence of expression of the Ad genome is limited in immunocompetent hosts by cellular immunity directed against the gene product of the vector. While most attention has been focused on cytotoxic T lymphocytes (CTL) directed against the low-level early and late Ad gene expression in the Ad vector-infected target cells, significant cellular immunity is likely also directed against the product of heterologous transgenes. To evaluate this concept, in vivo generation of CTL was evaluated in C57B1/6 and BALB/c mice with Ad vectors expressing a variety of heterologous transgenes, including Escherichia coli chloramphenicol acetyl transferase (CAT), beta-galactosidase (beta-Gal), cytosine deaminase, and human thrombopoietin (hTPO), with an Ad vector expressing no transgene ("null") as a control. Following intravenous administration of Ad vectors, spleen cells were harvested 2 weeks later, stimulated for 5 days with syngeneic cells infected with various Ad vectors, and then evaluated for CTL activity using 51Cr-release from syngeneic Ad vector-infected targets. In all cases, CTL directed against the heterologous transgene products was observed, although there were differences in the amounts of transgene-specific CTL. CTL directed against the transgene were also observed with other routes of administration, including intratracheal, subcutaneous, and intraperitoneal administration. These observations suggest that inclusion of a heterologous transgene in Ad vectors enhances the elimination of vector-infected cells, a circumstance that will be partially circumvented using autologous genes. For some applications, specific immune responses to products of transgenes delivered by Ad vectors might be exploited for therapeutic purposes.

Evidence that translation reinitiation abrogates nonsense-mediated mRNA decay in mammalian cells.

Nonsense codons upstream of and including position 192 of the human gene for triosephosphate isomerase (TPI) have been found to reduce the abundance of TPI mRNA to approximately 25% of normal. The reduction is due to the decay of newly synthesized TPI mRNA that co-purifies with nuclei. TPI mRNA that co-purifies with cytoplasm is immune to nonsense-mediated decay. Until now, a nonsense codon at position 23 has been the 5'-most nonsense codon that has been analyzed. Here, we provide evidence that a nonsense codon at position 1, 2 or 10 reduces the abundance of nucleus-associated TPI mRNA to an average of only 84% of normal because translation reinitiates at the methionine codon at position 14. First, converting codon 14 to one for valine increased the effectiveness with which an upstream nonsense codon reduces mRNA abundance. Second, when TPI gene sequences, including codon 14, were fused upstream of and in-frame to the translational reading frame of an Escherichia coli chloramphenicol acetyl transferase (CAT) gene that lacked an initiation codon, a nonsense codon at TPI position 1 or 2 allowed for the production of TPI-CAT that was an estimated 14 amino acids smaller than TPI-CAT produced by a nonsense-free gene, whereas a nonsense codon at TPI position 23 precluded the production of TPI-CAT. These and related findings lend credence to the concept that the nonsense-mediated reduction in the half-life of nucleus-associated TPI mRNA involves cytoplasmic ribosomes.

Developmental control of transcription of the cat reporter gene by a truncated mouse alphafetoprotein gene regulatory region in transgenic mice

A truncated mouse alphafetoprotein (AFP) gene promoter/enhancer region was tested for its ability to regulate the expression of the Escherichia coli chloramphenicol acetyltransferase (CAT) reporter gene in the livers of transgenic mice. The AFP regulatory region lacked any AFP gene structural DNA, included one enhancer sequence together with the proximal promoter sequence, and an element believed to be responsible for the postnatal repression of AFP gene transcription. The neonatal livers of AFP/CAT transgenic mice showed a high level of CAT enzyme expression, which was dramatically reduced between 7 and 14 days after birth. The staining of liver sections with anti-CAT antibodies showed that this expression was limited to hepatocytes. In one lineage, reexpression of CAT in the adult liver could be achieved by restitutive proliferation of hepatocytes following partial hepatectomy or CCl4-induced necrosis; reexpression in young animals (3-4 weeks of age) was even greater. These studies show that a truncated AFP promoter/enhancer region functions in a tissue-specific and developmental stage-specific fashion, and may be used to control the expression of other genes in the livers of transgenic mice.

Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene.

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5'-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding beta-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EcRE) was localized at position -75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

Precise quantitation of chloramphenicol acetyl transferase reporter mRNA by competitive polymerase chain reaction.

A strategy, based on competitive polymerase chain reaction (PCR) after reverse transcription, was developed to quantitate mRNA of the E. coli chloramphenicol acetyl transferase (CAT) gene. Precise quantitation of this rare mRNA is achieved by co-amplification of a constant amount of the complementary DNA (cDNA) with successive dilutions of a competitive DNA template of known concentration and with the use of the same primers. The unique EcoRI site located in the CAT coding sequence has been abolished in the competitive DNA. The two oligonucleotide primers used are both located in the CAT coding sequence at equal distance from the EcoRI site. After amplification, the PCR products from the cDNA to be quantified and from the competitor DNA are discriminated by EcoRI digestion, followed by separation of the resulting fragments by agarose gel electrophoresis. DNA amplified from the target cDNA is the only DNA digested by EcoRI into two fragments of identical size co-migrating in an agarose gel. After ethidium bromide staining, comparison of the intensity of fluorescence of the resulting bands in a competition series permits us to estimate precisely the amount of CAT cDNA and therefore of the mRNA to be quantified.

Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

Regulated expression of a mammalian nonsense suppressor tRNA gene in vivo and in vitro using the lac operator/repressor system.

We have exploited the Escherichia coli lac operator/repressor system as a means to regulate the expression of a mammalian tRNA gene in vivo and in vitro. An oligonucleotide containing a lac operator (lacO) site was cloned immediately upstream of a human serine amber suppressor (Su+) tRNA gene. Insertion of a single lac repressor binding site at position -1 or -32 relative to the coding region had no effect on the amount of functional tRNA made in vivo, as measured by suppression of a nonsense mutation in the E. coli chloramphenicol acetyltransferase gene following cotransfection of mammalian cells. Inclusion of a plasmid expressing the lac repressor in the transfections resulted in 75 to 98% inhibition of suppression activity of lac operator-linked tRNA genes but had no effect on expression of the wild-type gene. Inhibition could be quantitatively relieved with the allosteric inducer isopropylthio-beta-D-galactoside (IPTG). Similarly, transcription in vitro of lac operator-linked tRNA genes in HeLa cell extracts was repressed in the presence of lac repressor, and this inhibition was reversible with IPTG. These results demonstrate that the bacterial lac operator/repressor system can be used to reversibly control the expression of mammalian genes that are transcribed by RNA polymerase III.

E1A-responsive elements for repression of rat fibronectin gene transcription.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

E1A-responsive elements for repression of rat fibronectin gene transcription.

The level of fibronectin (FN) gene transcription in resting rat 3Y1 cells is very high but decreases steeply after growth stimulation by serum or by the induction of E1A expression. To study the mechanism of this E1A-mediated down-regulation, the 5' flanking regions of the FN gene with various deletions and substitutions were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and introduced into resting 3Y1 cells with E1A expression plasmids. The results indicate that the G10 stretch located from nucleotide position -239 to -230 and two GC boxes from position -105 to -95 and position -54 to -44 are the primary E1A-responsive elements for repression of the FN gene. Two GC boxes also contain a G10 stretch that is interrupted by the presence of an internal C residue. These sequences overlap with the Sp1 motif GGGCGG. Substitution of the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, completely abolished the E1A sensitivity of the promoter. Analysis of the E1A domains by using various E1A deletion mutants indicated that the domain for binding to the retinoblastoma susceptibility gene product (RB) is essential for efficient repression. These results suggest that the gene encoding a negative factor(s) binding to the three G-rich sequences in the FN promoter is repressed by RB in resting 3Y1 cells and derepressed by expression of E1A.

Retroviral vectors containing putative internal ribosome entry sites: development of a polycistronic gene transfer system and applications to human gene therapy.

Recombinant retroviral vectors producing multicistronic mRNAs were constructed. Picornavirus putative internal ribosome entry sites (IRES) were used to confer cap-independent translation of an internal cistron. Internal cistrons were engineered by ligation of various lengths of the IRES of encephalomyocarditis (EMC) virus or polio virus to the E. coli chloramphenicol acetyltransferase (CAT) gene. The IRES/CAT fusions were introduced into retroviral vectors 3' to the translation stop codon of the neomycin phosphotransferase (NEO) gene, and the molecular constructs transfected into retroviral vector packaging lines. Retroviral vector producer cells efficiently express the internal CAT gene product only when the full length IRES is used. Both the EMC/CAT and polio/CAT retroviral vectors produced high titer vector supernatant capable of productive transduction of target cells. To test the generality of this gene transfer system, a retroviral vector containing an IRES fusion to the human adenosine deaminase (ADA) gene was constructed. Producer cell supernatant was used to transduce NIH/3T3 cells, and transduced cells were shown to express NEO, and ADA. Novel three-gene-containing retroviral vectors were constructed by introducing the EMC/ADA fusion into either an existing internal-promoter-containing vector, or a polio/CAT bicistronic vector. Producer cell clones of the three-gene vectors synthesize all three gene products, were of high titer, and could productively transduce NIH/3T3 cells. By utilizing cap-independent translation units, IRES vectors can produce polycistronic mRNAs which enhance the ability of retroviral-mediated gene transfer to engineer cells to produce multiple foreign proteins.

Improved Encapsulation of DNA in pH-Sensitive Liposomes for Transfection

AbstractA simple method has been developed to prepare liposomes containing large amounts of DNA. The procedure consisted of three cycles of freeze-thawing a mixture of sonicated liposomes and DNA. The encapsulation efficiency depended on the size of DNA. For a small plasmid (2.7 kb), approximately 40% of input DNA was entrapped with an efficiency of 16 μgDNA/μmol lipid. For larger plasmids, the encapsulation efficiency decreased considerably. Transfection of cultured mouse L929 cells mediated by the DNA-containing liposomes was assayed with a plasmid containing the E. coli chloramphenicol acetyl transferase gene. The transfection activity of the liposome was primarily determined by its pH sensitivity. Acid-sensitive liposomes transfected cells efficiently, whereas pH-insensitive liposomes were much less active. The level of the expression of the exogenous gene in the treated cells could be further modulated by protein kinase C (PKC) activators that were incorporated into the liposomal membrane as a minor ...

Mammalian liver contains an activity which mimics bacterial chloramphenicol acetyltransferase

Rat liver extracts contain an activity which mimics Escherichia coli chloramphenicol acetyltransferase (CAT); the latter is commonly used to report transcriptional activation of chimeric genes transfected into cultured cells. Although the activities are indistinguishable by the standard thin-layer chromatography assay, alternate methods can discriminate between them. The rat CAT-like activity appears to be an integral membrane protein. It was observed in the microsomal fraction of both liver and kidney. Similarly CAT-like activities were detected in mouse, rabbit and pig liver. In addition, liver homogenates which contain the CAT-like activity also contain a heat-labile inhibitor of (authentic) bacterial CAT.

Chloramphenicol acetyltransferase expression in marine Rhodobacter sp. NKPB 0021 by use of shuttle vectors containing the minimal replicon of an endogenous plasmid

A vector, pUK318, was constructed to allow the expression of foreign genes in the marine photosynthetic bacterium Rhodobacter sp. NKPB 0021. This strain has been cured of its two endogenous plasmids pUK318 consists of a 2.3-kb PstI-BamHI restriction fragment, containing a marine Rhodobacter plasmid replication region, cloned into pUC18. This fragment was derived from plasmid pRD31, a 3.1-kb endogenous plasmid purified from the marine strain Rhodobacter sp. NKPB 043402. A kanamycin resistance gene from Tn 903 was cloned into the PstI restriction site to provide antibiotic selection. pUK318 was transferred to Rhodobacter sp. NKPB 0021 by transformation, and efficiencies of 7.2 × 10 −5 were obtained. Furthermore, pUK318 was stably maintained when transformants were grown either heterotrophically or photosynthetically in the absence of antibiotics. pUK318 was used to express the Escherichia coli chloramphenicol acetyl transferase (CAT) gene in Rb. NKPB 0021. Transformants expressed a maximum CAT activity of 1.12 mmol/min/g dry cells. In addition, the DNA region essential for pUK318 replication in Rb. NKPB 0021 was localized to a 1.36-kb HincII-PstI fragment. This is the first report of a plasmid vector containing a marine Rhodobacter-specific replicon that allows stable expression of foreign genes in the absence of antibiotic selection.

Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells

In this study, we demonstrated that lipofectin-treated DNAs which were injected into mouse brain could be incorporated and expressed by brain cells. When L7RH-βgal plasmid DNA harboring E. coli β-galacto-sidase gene fused with the nuclear location signal of SV40 T-antigen gene was injected into brains of 1-week-old mice, cells whose nuclei appeared to be densely stained with the chromogenic substrate X-gal were detected in several portions of the brain till 9 days after injection. Injection of pMLV-CAT plasmid DNA which contains the E. coli chloramphenicol acetyltransferase (CAT) gene also resulted in cells immunoreactive to the anti-CAT antibody.

Expression of a Bacterial Gene in a Trypanosomatid Protozoan

A simple and reproducible assay for DNA-mediated transfection in the trypanosomatid protozoan Leptomonas seymouri has been developed. The assay is based on expression of the Escherichia coli chloramphenicol acetyl transferase (CAT) gene flanked by Leptomonas DNA fragments that are likely to contain necessary elements for gene expression in trypanosomes. After electroporation of cells in the presence of plasmid DNA, CAT activity was detected in crude cell lysates. No activity was detected when the orientation of the L. seymouri mini-exon sequence (placed upstream of the CAT gene) was reversed, or in additional control experiments. This system provides a method for defining transcriptional control elements in trypanosomes.

Identification of sequence requirements and trans-acting functions necessary for regulated expression of a human cytomegalovirus early gene.

We analyzed the regulation of expression of a human cytomegalovirus (HCMV) early transcription unit encoded by EcoRI fragments R and d (map units, 0.682 to 0.713), located within the long unique segment of the genome. This region specified a 2.2-kilobase class of spliced transcripts which encode several related proteins. To define important upstream regulatory elements of this gene, we generated hybrid plasmids in which 5'-promoter sequences were fused to the Escherichia coli chloramphenicol acetyltransferase (CAT) gene and tested these hybrid genes in transient expression assays in human fibroblast cells. The stimulation of CAT activity in HCMV-infected cells was found to reflect an induction of correctly initiated hybrid mRNA, which was dependent on the de novo synthesis of some virally induced factor(s). A time course experiment showed the correct early kinetics of CAT expression. Analysis of a series of 5'-promoter deletion plasmids, ending between -323 and -7 base pairs relative to the transcription start site, showed a stepwise reduction in inducible CAT activity, suggesting that this HCMV early promoter consists of multiple elements. One of these elements resembles the binding site of a previously identified cellular "transcription" factor. We also examined the role of specific virus-encoded factors in the transactivation of this promoter. Cotransfection of human fibroblasts with the 2.2-kilobase RNA promoter-CAT construct and plasmids containing different immediate-early genes showed that expression of CAT from this promoter was stimulated by the region of the HCMV genome encoding the immediate-early 1 and 2 gene products.