Simian Virus 40 Early Promoter Research Articles

Weak promoters to drive selection marker expression: Improvement of cell line development process for therapeutic protein production in CHO-K1 cells.

Chinese Hamster Ovary cells have been widely used as host cells for production of recombinant therapeutic molecules. Cell line development is a decisive step, which must be carried out with an efficient process. In particular, degree of selection stringency is an important parameter for identification of rare, high-producing cell lines. In the CHOZN® CHO K1 platform, selection of top-producing clones is based on puromycin resistance, whose expression is driven by Simian Virus 40 Early (SV40E) promoter. In this study, novel promoters have been identified to drive expression of selection marker. Decrease of transcriptional activity compared to SV40E promoter was confirmed by RT-qPCR. Selection stringency was increased, as seen by decreased surviving rate of transfected mini-pools and longer recovery duration of transfected bulk pools. Several promoters led to a 1.5-fold increase of maximum titer and a 1.3-fold increase of mean specific productivity of the monoclonal antibody over the clone generation. Expression level was maintained stable over long term cultivation. Finally, productivity increase was confirmed on several monoclonal antibodies and fusion proteins. Lowering the strength of promoter for expression of selective pressure resistance is an efficient strategy to increase selection stringency, which can be applied on industrial CHO-based cell line development platforms.

The Activity of SV40 Promoter Can Be Inhibited by Overexpression of Heme Oxygenase-1 in Tumor Cells

Heme oxygenase-1 (HO-1) is both beneficial and detrimental to the host in some viral infections by catalyzing the conversion of heme to biliverdin, iron, and carbon monoxide. Simian Virus 40 (SV40) early promoter plays an important role in transforming many cells as it can drive the transcription of large T antigen, which is a potent oncogene. In order to determine the effect of HO-1 on the SV40 early promoter, tumor cells overexpressing HO-1 and HO-1 dominant-negative mutant (glycine143 mutated to histidine) (HO-1G143H) were used. Western blot and HO activity for HO-1/HO-1G143H expression, cell growth, and luciferase activity driven by SV40 promoter were detected in this study. The luciferase activity was suppressed notably in BGC-823 cells transiently overexpressing HO-1, but significantly increased in BGC-823 cells transiently overexpressing HO-1G143H, compared with the mock, respectively. HO-1 overexpression in BGC-823 cells caused the cells containing Blasticidin-resistant gene driven by SV40 promoter to grow slowly under Blasticidin screening, compared with control groups. The luciferase activities were also suppressed in BGC-823, A549, and HepG2 cells stably overexpressing HO-1, and increased in these cell lines stably overexpressing HO-1G143H, compared with the mock, respectively. The results demonstrated that overexpression of HO-1 suppressed transcription driven by SV40 promoter in tumor cells and that HO-1 catalysates might play a major role in the process. Our preliminary results suggested that HO-1 might possess promising counteraction in cell transformation by suppressing SV40 large T-antigen expression, potentially applicable to therapeutic interventions in some virus diseases.

A Sequence Motif in the Simian Virus 40 (SV40) Early Core Promoter Affects Alternative Splicing of Transcribed mRNA

To identify new sequence elements in the promoter that affect splicing patterns of pre-mRNAs, we analyzed effects of different promoters on alternative splicing of model reporter genes. We compared the E1a alternative splicing pattern in transcripts expressed from the full-length cytomegalovirus, SV40 early, or a hybrid cytomegalovirus/SV40 early promoter and found that the hybrid promoter improved selection of the suboptimal E1a 5'SS-1. Expressing RNA from the hybrid promoter also enhanced selection of suboptimal splice sites in other alternatively spliced reporter genes, demonstrating the generality of this effect. Unlike previously defined promoter elements shown to affect alternative splicing, which were located in the enhancer/upstream activating sequences, the motif identified in this work is positioned within the core promoter; it is comprised of eight T-residues directly upstream of the SV40 early TATA box. This motif was previously implicated in DNA bending and negative regulation of transcription. Together, these results suggest that the identity of transcription complex assembled in the core promoter-dependent fashion can affect splice site selection during pre-mRNA splicing, perhaps by influencing the processivity of transcription elongation.

Enhancing and suppressing effects of dexamethasone on transgene expression in vitro

Gene therapy is becoming an important treatment modality against various diseases including cancer, genetic disorders, infectious diseases and inflammatory diseases. For achieving successful gene therapy, it is extremely important to control the expression of a transduced therapeutic gene. However, there have been few studies examining the effect of glucocorticoid on transgene expression. We demonstrate here that the synthetic glucocorticoid dexamethasone significantly affected transgene expression in vitro. Bone marrow cells freshly prepared from rats and murine fibroblasts were infected with retroviruses carrying the neomycin phosphotransferase gene under the transcriptional control of the retroviral long terminal repeat promoter and the reporter lacZ gene under the transcriptional control of the simian virus 40 (SV40) early promoter. Retrovirus-infected cells were selected by G418. When retrovirus-infected bone marrow cells were cultured in the presence of dexamethasone, lacZ expression was markedly decreased. This suppressive effect of dexamethasone on transgene expression in bone marrow cells appeared to be mediated by a methylation-independent mechanism, because the suppressive effect was also observed in bone marrow cells that were supposed to contain a considerable number of methylated cells. In marked contrast, when retrovirus-infected fibroblasts were cultured in the presence of dexamethasone, lacZ expression was significantly increased. The enhancing and suppressing effects of dexamethasone on transgene expression were considered to be independent on promoter types, because the SV40 early promoter used to control lacZ expression does not contain a glucocorticoid-responsive element. Therefore, the different effects of dexamethasone on transgene expression appeared to be dependent on cell types. These results indicate that dexamethasone may play an important role for achieving successful gene therapy. Furthermore, these observations may also have important implications for future clinical applications of gene therapy.

A novel approach for inducing enhanced and selective transgene expression in hepatocellular‐carcinoma cells

Utility of the α-fetoprotein (afp) promoter for gene therapy against hepatocellular carcinoma (HCC) is limited because of the weak promoter activity. To circumvent this, the 5.1-kb 5`-flanking sequence of the human afp gene including the entire enhancer and silencer regions as well as the promoter region was employed for achieving strong, HCC-selective transgene expression. To thoroughly inhibit the promoter activity of the 5`-flanking sequence of the human afp gene, the afp 5`-flanking region was inserted downstream of the human interleukin-2 (il-2) gene controlled by the simian-virus-40 (SV40) early promoter. il-2-production ability of HCC cells transduced with the construct was significantly enhanced compared with that transduced with the same construct lacking the afp 5`-flanking region. Importantly, il-2 production of non-HCC cells was substantially inhibited by the addition of the afp 5`-flanking region to the construct. When the afp 5`-flanking region was inserted downstream of the human tumor-necrosis-factor-α (tnf-α) gene controlled by the retrovirus long-terminal-repeat (LTR) enhancer/promoter, tnf-α production ability of HCC cells was significantly enhanced and that of non-HCC cells was significantly suppressed compared with that transduced with the same construct lacking the afp 5`-flanking region. Our results indicated that the afp 5`-flanking region gave the enhanced HCC-selective activity to the non-tissue specific SV40 early promoter and LTR enhancer/promoter. It is essential for successful gene therapy to induce strong, target-cell-selective transgene expression. This novel strategy, therefore, may contribute to the establishment of HCC-selective cancer gene therapy. Int. J. Cancer 87:247–252, 2000. © 2000 Wiley-Liss, Inc.

A novel approach for inducing enhanced and selective transgene expression in hepatocellular-carcinoma cells

Utility of the alpha-fetoprotein (afp) promoter for gene therapy against hepatocellular carcinoma (HCC) is limited because of the weak promoter activity. To circumvent this, the 5.1-kb 5;-flanking sequence of the human afp gene including the entire enhancer and silencer regions as well as the promoter region was employed for achieving strong, HCC-selective transgene expression. To thoroughly inhibit the promoter activity of the 5;-flanking sequence of the human afp gene, the afp 5;-flanking region was inserted downstream of the human interleukin-2 (il-2) gene controlled by the simian-virus-40 (SV40) early promoter. il-2-production ability of HCC cells transduced with the construct was significantly enhanced compared with that transduced with the same construct lacking the afp 5;-flanking region. Importantly, il-2 production of non-HCC cells was substantially inhibited by the addition of the afp 5;-flanking region to the construct. When the afp 5;-flanking region was inserted downstream of the human tumor-necrosis-factor-alpha (tnf-alpha) gene controlled by the retrovirus long-terminal-repeat (LTR) enhancer/promoter, tnf-alpha production ability of HCC cells was significantly enhanced and that of non-HCC cells was significantly suppressed compared with that transduced with the same construct lacking the afp 5;-flanking region. Our results indicated that the afp 5;-flanking region gave the enhanced HCC-selective activity to the non-tissue specific SV40 early promoter and LTR enhancer/promoter. It is essential for successful gene therapy to induce strong, target-cell-selective transgene expression. This novel strategy, therefore, may contribute to the establishment of HCC-selective cancer gene therapy.

Characterization and evaluation of detoxification functions of a nontumorigenic immortalized porcine hepatocyte cell line (HepLiu).

Primary porcine hepatocytes (PPH) are currently used in research and therapeutic applications as the biological component of extracorporeal liver assist devices to overcome the shortage of human hepatocytes. However, their finite life span and typically rapid loss of functions limit their utility. An immortalized, nontumorigenic, highly differentiated porcine hepatocyte cell line was developed in our laboratory to resolve these disadvantages. PPH were transfected with simian virus 40 (SV40) T antigen under the control of the SV40 early promoter. From the established 69 clones, 23 clones displaying hepatocyte-like morphology were screened for diazepam metabolism. One clone, HepLiu D63, has been maintained in culture for > 2 years, through more than 60 passages and 240 divisions. Albumin protein, present in early passages, was lost at later passages, but albumin transcript still was detectable in later passages. Carbamoyl phosphate synthetase, a gateway enzyme of the urea cycle, was consistently detectable in HepLiu cells. Cytokeratin 18, a characteristic marker of primary hepatocytes, was detected by both immunofluorescent staining and Western blot in HepLiu cells. Furthermore, maintenance of P450 functions in HepLiu cells was evidenced by diazepam and 7-ethoxycoumarin metabolites measured by HPLC. Phase II conjugative function was measured as acetaminophen glucuronidation. P450 dealkylase was demonstrated microscopically by the conversion of a nonfluorescent substrate to a fluorescent product. Both Northern blot analysis and immunofluorescent staining showed SV40 T antigen expression in the nuclei of HepLiu cells. No tumor formation occurred when HepLiu cells were injected into severe combined immunodeficient (SCID) mice nor was the TAI (a tumor marker) mRNA expressed, even in later passages. This immortalized, nontumorigenic, highly functional cell line may provide a valuable tool for drug/toxicological studies, liver biologic regulation studies, and artificial liver support systems.

Use of SV40 to immunize against hepatitis B surface antigen: implications for the use of SV40 for gene transduction and its use as an immunizing agent.

We have described a novel gene transfer system, in which replication-incompetent, T antigen-deleted simian virus-40 (SV40) is used as the transduction vehicle. We report here successful immunization using such an SV40-derived viral vector. Hepatitis B surface antigen (HBsAg) cDNA was cloned downstream of two tandem SV40 early promoters to yield a T antigen-deficient SV40 derivative, SV(HBS). Cultured TC7 cells were exposed to SV(HBS), and expression of HBsAg was detected 24 h later by Northern blot and RT-PCR analysis. Immunochemistry and Western blot analysis were also performed 24 h after infection to detect expression of HBsAg. Once it was ascertained that we could express HBsAg in this way, we used SV(HBS) to elicit anti-HBs. SV(HBS) was injected intraperitoneally or subcutaneously into mice every 4 weeks. These mice were bled every 2 weeks and their sera assayed for antibody activity against HBsAg and SV40. Production of anti-HBs was measured by ELISA and confirmed by Western blot analysis, both of which demonstrated significant levels of anti-HBs after the second injection. We also tested production of anti-SV40 antibodies by the ability of sera to neutralize SV(HBS) infectivity. We found no evidence of neutralization of SV(HBS) infectivity even after eight inoculations. Thus, replication-incompetent SV40 is itself not a strong antigen. Our data suggest that SV40-based transduction systems may be a useful vehicle for immunization and for other gene transfer applications when a need for multiple inoculations is anticipated.

Suppression of Gene Expression on the Simian Virus 40 Major Late Promoter by Human TR4 Orphan Receptor: A MEMBER OF THE STEROID RECEPTOR SUPERFAMILY

The key expression of the simian virus 40 (SV40) major late promoter could be repressed by the human TR4 orphan receptor via the +55 region of the SV40 major late promoter (nucleotide numbers 368-389, 5' -GTTA-AGGTTCGTAGGTCATGGA-3'). Using the coupled in vitro transcribed and translated TR4 orphan receptor with a molecular mass of 67.3 kilodaltons, electrophoretic mobility shift assay showed specific binding with a dissociation constant of 1.09 nM between the TR4 orphan receptor and the SV40 +55 oligonucleotides. In addition, chloramphenicol acetyltransferase assay demonstrated that this SV40 +55 region can function as a repressor via the TR4 orphan receptor, suppressing the transcriptional activities of both SV40 early and late promoters. Together, our data suggest that the TR4 orphan receptor may play an important role for the suppression of the SV40 gene expression.

Relationship between expression of epidermal growth factor and simian virus 40 T antigen in a line of transgenic mice

The pattern of expression of the simian virus 40 (SV40) T antigen gene and resultant dysplasia were re-examined in a line of transgenic mice in which the T antigen gene was under the control of the SV40 early promoter. We found that T antigen expression in the kidney, and resulting dysplastic lesions, occurred exclusively in the distal convoluted tubules and the ascending limbs of Henle. Epidermal growth factor (EGF) expression in the kidney of normal mice was similarly immunolocalized. The correlation between high EGF immunoreactivity in normal mouse tissues and T antigen expression in the transgenic counterpart was also seen in the choroid plexus epithelium and in the submandibular glands of male mice. T antigen was not found in the submandibular gland of transgenic females. Similarly, EGF was only rarely detected in the normal female submandibular gland. In contrast to the correlation between T antigen expression in the transgenic mice and EGF expression in the corresponding tissues of the normal mice, within the dysplastic lesions of the transgenic mice EGF expression was severely diminished. Adenocarcinomas of the male submandibular gland from another line of transgenic mice that expresses the Int-1 transgene, showed similarly reduced levels of immunostaining for EGF. Thus, reduced expression of EGF might be a general feature of dysplasia and tumorigenesis in those tissues that normally express EGF.

Identification of Human TR2 Orphan Receptor Response Element in the Transcriptional Initiation Site of the Simian Virus 40 Major Late Promoter

A DNA response element (TR2RE-SV40) for the TR2 orphan receptor, a member of the steroid-thyroid hormone receptor superfamily, has been identified in the simian virus 40 (SV40) +55 region (nucleotide numbers 368-389, 5'-GTTAAGGTTCGTAGGTCATGGA-3'). Electrophoretic mobility shift assay, using in vitro translated TR2 orphan receptor with a molecular mass of 67 kilodaltons, showed a specific binding with high affinity (dissociation constant = 9 nM) for this DNA sequence. DNA-swap experiments using chloramphenicol acetyl-transferase assay demonstrated that androgen can suppress the transcriptional activities of SV40 early promoter via the interaction between this TR2RE-SV40 and the chimeric receptor AR/TR2/AR with the DNA-binding domain of the TR2 orphan receptor flanked by the N-terminal and androgen-binding domains of the androgen receptor. In addition, this TR2RE-SV40 can function as a repressor to suppress the transcriptional activities of both SV40 early and late promoters. Together, these data suggest the TR2RE-SV40 may represent the first identified natural DNA response element for the TR2 orphan receptor that may function as a repressor for the SV40 gene expression.

Nucleosome binding by the constitutive transcription factor Sp1.

The constitutive transcription factor Sp1 plays a role in the transcription of numerous viral and cellular genes including constitutive "housekeeping" genes and inducible genes. Sp1 has also been implicated in the formation of a nucleosome-free region in Simian virus 40 (SV40) minichromosomes. To investigate the potential functions of Sp1 in remodeling chromatin structures, Sp1 was analyzed for the ability to bind its recognition sites (GC boxes) in DNA fragments reconstituted into nucleosome cores. Sp1 was found to bind to the GC boxes of the SV40 early promoter when this element was reconstituted into nucleosome cores. The affinity of Sp1 for the nucleosomal SV40 early promoter DNA was reduced approximately 10-20-fold relative to naked DNA. A peptide containing only the zinc fingers of Sp1 was also capable of binding nucleosomal DNA, indicating that the glutamine-rich and serine/threonine-rich domains of Sp1 are not required for nucleosome binding. The binding of Sp1 to nucleosome cores resulted in the formation of a ternary Sp1-nucleosome complex.

Conditionally immortalized intestinal epithelial cells: novel approach for study of differentiated enterocytes

Clonal cell lines have been established from primary fetal rat intestinal epithelial cells by stable transfection with plasmids containing either the simian virus 40 (SV40) large T-antigen gene under the control of the heavy metal inducible metallothionein promoter (pMTWt) or the thermolabile SV40 T-antigen gene under the control of the SV40 early promoter (pZipSVtsa58). pMTWt-transfected cells produced sufficient T-antigen to allow them to proliferate both when the metallothionein promoter was induced and uninduced. No differences were observed in the pattern of intestinal epithelial markers expressed when the cells were cultured in the presence or absence of inducing agent (zinc). In contrast, fetal rat intestinal epithelial cells transfected with pZipSVtsa58 were immortalized conditionally; cells proliferated at 32 degrees C but ceased to proliferate between 48 and 72 h of culture at 39 degrees C. Four of these cell lines were characterized in detail; they showed microvilli and tight junctions as well as dome formation and expressed functional and biochemical markers of intestinal epithelial cells, including keratins 8, 19, and 21, aminopeptidase N, and dipeptidyl peptidase IV. One cell line, 2/4/A1, expressed in addition a low level of lactase and sucrase-isomaltase. The amount and/or activity of some of these markers changed during the switch from the proliferative to the nonproliferative state (switch from culture at 32 to 39 degrees C), resulting in a more differentiated phenotype and mimicking similar changes taking place during intestinal epithelial cell differentiation in vivo.

Genetic definition of a new bovine papillomavirus type 1 open reading frame, E5B, that encodes a hydrophobic protein involved in altering host-cell protein processing

We have previously observed that bovine papillomavirus type 1 (BPV-1) induces the appearance of five cellular proteins in C127 mouse fibroblasts, four of which appear to arise by altered processing of resident endoplasmic reticulum proteins. Studies of various cell lines revealed that expression of the 3' end of the BPV early region was sufficient for induction of these changes. To identify the BPV gene responsible, we have utilized the simian virus 40 (SV40)/BPV-1 recombinant virus Pava-1, which expresses the 3' end of the BPV early region behind an SV40 early promoter. C127 cells infected with Pava-1 for 48 h show the expected BPV-associated alterations, as do cells infected with Pava constructs mutated in the E5 or E2 genes. However, a mutation in the start codon of a previously ignored open reading frame extending from nucleotides 4013 to 4170 (E5B) eliminated the BPV-associated changes. Similar results were obtained with COS cells infected with the Pava mutants and C127 cells transformed by full-length mutated BPV. Despite its influence on the processing of cellular endoplasmic reticulum proteins, this mutation in E5B did not alter BPV-transforming efficiency or the ability of transformants to form colonies in soft agar. The E5B open reading frame encodes a hydrophobic 52-amino-acid polypeptide that shares structural similarities with HPV6 E5A and HPV16 E5. Speculations on a role for E5B in the viral life cycle are discussed.

Phenotypic alterations in fibroblasts and fibrosarcoma cells that overexpress latent transforming growth factor-beta 1.

Mouse embryo-derived AKR-2B fibroblasts and murine fibrosarcoma cells (the 1591 cell line) were transfected with a murine transforming growth factor-beta 1 (TGF beta 1) cDNA under the transcriptional control of either the simian virus-40 early promoter or the cytomegalovirus promoter/enhancer. Selected clones secreted 2- to 4-fold more TGF beta-competing activity into their media than the parental cell line or neomycin-transfected controls. The TGF beta 1 released into the cell-conditioned medium was latent. Despite the latency of the overexpressed TGF beta 1, TGF beta 1-transfected cells exhibited phenotypic features of TGF beta 1-treated cells. When confluent, the TGF beta 1-transfected cells had the morphological characteristics of the parental cells that have been treated with active TGF beta 1. AKR-2B cells that expressed higher levels of TGF beta 1 also expressed high levels of c-sis and c-myc mRNAs and decreased TGF beta 2 and TGF beta 3 mRNAs in the same manner as parental AKR-2B cells that had been treated with active TGF beta 1. The transfected 1591 cells that overexpressed TGF beta 1 bound less [125I]TGF beta 1 than did parental 1591 cells, but after a mild acid wash demonstrated an increase in [125I]TGF beta 1 binding. Our results suggest that these TGF beta 1-transfected fibroblast and fibrosarcoma cells have the capacity to activate TGF beta; however, as very little activated TGF beta is detected in the medium, it is hypothesized that these cells activate latent TGF beta 1 and bind the activated TGF beta 1, thus acquiring a phenotype consistent with TGF beta 1-treated cells.

In vitro initiation of transcription by RNA polymerase II on in vivo-assembled chromatin templates.

We have studied the initiation of transcription in vitro by RNA polymerase II on simian virus 40 (SV40) minichromosomal templates isolated from infected cells. The efficiency and pattern of transcription from the chromatin templates were compared with those from viral DNA templates by using two in vitro transcription systems, either HeLa whole-cell extract or basal transcription factors, RNA polymerase II, and one of two SV40 promoter-binding transcription factors, LSF and Sp1. Dramatic increases in numbers of transcripts upon addition of transcription extract and different patterns of usage of the multiple SV40 initiation sites upon addition of Sp1 versus LSF strongly suggested that transcripts were being initiated from the minichromosomal templates in vitro. That the majority of transcripts from the minichromosomes were due to initiation de novo was demonstrated by the efficient transcription observed in the presence of alpha-amanitin, which inhibited minichromosome-associated RNA polymerase II, and an alpha-amanitin-resistant RNA polymerase II, which initiated transcription in vitro. The pattern of transcription from the SV40 late and early promoters on the minichromosomal templates was similar to the in vivo pattern of transcription during the late stages of viral infection and was distinct from the pattern of transcription generated from viral DNA in vitro. In particular, the late promoter of the minichromosomal templates was transcribed with high efficiency, similar to viral DNA templates, while the early-early promoter of the minichromosomal templates was inhibited 10- to 15-fold. Finally, the number of minichromosomes competent to initiate transcription in vitro exceeded the amount actively being transcribed in vivo.

Involvement of simian virus 40 (SV40) small t antigen in trans activation of SV40 early and late promoters.

We have previously found that simian virus 40 (SV40) small t antigen (small t) can trans activate the E2A and VA-I genes of adenovirus in plasmid DNA-transfected cells (M. R. Loeken, I. Bikel, D. M. Livingston, and J. Brady, Cell 55:1171-1177, 1988). To determine whether trans activation by small t might be involved in the SV40 productive infection cycle, we examined the effects of cotransfecting plasmids encoding small t with plasmids containing the chloramphenicol acetyltransferase (CAT) gene linked to the SV40 early or late promoter. Small t increased three- to fivefold the expression of a CAT plasmid linked to the SV40 early promoter and enhancer. Small t expression had no effect by itself on CAT activity directed by the SV40 late promoter, but small t enhanced the effect of a suboptimal concentration of a plasmid expressing large T up to 10-fold. When the concentration of the plasmid expressing large T was increased to a level at which large T alone stimulated the late promoter ninefold, the enhancement by small t was only twofold. The effects of small t on both the SV40 early and late promoters depended on sequences within the small t-unique domain, since a plasmid expressing only the first 82 amino acids common to both large T and small t was inactive. The effects of small t on early- and late-promoter-directed CAT enzyme activity was reflected in increased CAT mRNA as measured by S1 analysis. These results suggest that SV40 small t may play a role in viral infection by increasing transcription from the early promoter and from the late promoter at times when large T levels are low.

Simian virus 40 small-t does not transactivate RNA polymerase II promoters in virus infections

Transcriptional stimulatory properties of virus-encoded transactivators appear to be critical for viral gene expression and may be linked to cellular transformation in certain cases. Recently, the simian virus 40 (SV40) 17-kDa small-t antigen was shown to stimulate transcription of polymerase II and III genes in transient transfection assays. In experiments performed in our laboratory, two of the polymerase II promoters of the adenovirus genome, namely, the EII-early and EIII promoters, were transactivation, we examined the transient transfection assays. To further elucidate the mechanism of this transactivation, we examined the ability of small-t to transactivate the adenovirus type 5 EII-early and EIII promoters in CV-1 cells under conditions in which the small-t gene or the reporter genes were introduced into the cells through transfection and other routes. In one approach, we used established CV-1 cell lines which constitutively express the small-t gene, and study of the EII-early promoter was afforded by infection of an EIA-negative adenovirus type 5 variant. For the second approach, a recombinant adenovirus was constructed in which small-t was expressed from a replication origin-negative SV40 early promoter in the EIA region of an adenovirus vector (Ad-SV-t). The effect of small-t on adenovirus EII-early and EIII promoter expression was studied in coinfection or single-infection experiments. In both cases, transcription of the adenovirus early promoters was not stimulated by small-t. These and other results indicate that transactivation of polymerase II promoters by small-t occurs only when the target gene is in a transiently transfected state. Thus, small-t-mediated transactivation of polymerase II promoters is dependent on the type of assay system used and may be mechanistically different from that of the widely studied EIA.

Regulation of the Uteroferrin Gene Promoter in Endometrial Cells: Interactions among Estrogen, Progesterone, and Prolactin*

Expression of the gene for the porcine transplacental iron transport protein uteroferrin (UF) is largely restricted to the uterus, where it is differentially regulated by estrogen (E) and progesterone (P). To study the regulatory mechanisms subserving these effects, a 2-kilobase genomic fragment corresponding to -2005 to 48 nucleotides of the UF gene was ligated up-stream to the reporter gene chloramphenicol acetyltransferase (CAT). This construct (UF-CAT) was transiently transfected into rabbit endometrial (HRE-H9), mouse fibroblastic (AKR-2B), and human choriocarcinoma (JEG-3) cells. The basal gene promoter activity of UF-CAT was exhibited in H9 cells, but not in AKR-2B or JEG-3 cells. In contrast, a simian virus-40 early promoter (SV2) was functional in all three cell lines. The H9 cells were used to examine steroid regulation of the UF gene promoter. The CAT expression in H9 cells primed with E and PRL, but not with E or PRL alone, was stimulated by P. In contrast, basal activity of SV2 in these cells was unaffected by hormones, singly or in combination. To examine the basis for the E/PRL-dependent response to P, levels of P and E receptors in H9 cells were quantified. PRL and E plus PRL increased the number of high affinity sites for P, but had little effect on levels of high affinity sites for E in treated vs. untreated H9 cells. In vivo administration of PRL to cyclic gilts had no effect on levels of endometrial UF mRNA and secreted UF protein; however, E- plus PRL-treated gilts had higher (P less than 0.05) levels of endometrial UF mRNA and luminal UF than PRL-treated gilts. These results demonstrate in vitro functional activity of the UF gene promoter and associated 5' flanking region and suggest that sequences within this region may mediate tissue-specific and steroid hormone-regulated expression of the UF gene. Moreover, interactions among E, PRL, and P modulate UF gene expression in vivo and in vitro.

Generation of mammalian cells expressing stably measles virus proteins via bicistronic RNA

The proteins of measles virus are believed to be cytotoxic, and have never been expressed stably from the cloned genes in cultured cells. We found that measles viral proteins can be expressed via a bicistronic RNA. The dominantly selectable DHFR ∗ protein-coding region encoding a mutant dihydrofolate reductase was inserted into the 3'-untranslated regions of the measles viral genes encoding nucleoprotein (N), matrix (M) protein, and hemagglutinin (H). The tandemly arranged cistrons were placed under control by the inducible promoter of human metallothionein II A gene, or the noninducible early promoter of simian virus 40. Upon transfecting into mammalian cells, these gene constructs synthesized bicistronic RNAs. The downstream DHFR ∗ gene conferred resistance to methotrexate (MTX). Cells that survived MTX selection expressed stably the N, M, or H protein of measles virus. Expression of N protein was further inducible by cadmium chloride treatment. This system will be useful for studying the protein functions of measles virus, and could be applied to express other potentially toxic gene products.