Levels Of Chloramphenicol Acetyltransferase Activity Research Articles

Hormonal and nutritional regulation of the M‐CPTI gene expression in a transgenic mice model in vivo

To study the molecular basis of tissue –specific and hormonally regulated expression of the muscle carnitine palmitoyltransferase I (M-CPTI) gene in vivo, we generated lines of transgenic mice carrying 1.1-kb of the 5′-flanking region (-1194 to +54) of the human heart M-CPTI gene fused to a chloramphenicol acetyltransferase (CAT) reporter gene. The reporter gene was strongly expressed in tissues that normally express high levels of M-CPTI mRNA, which include heart and skeletal muscle, but not in liver and kidney. We also examined the hormonal and nutritional regulation of the M-CPTI (1.1)-CAT reporter gene in the transgenic mice. When the mice were fasted for 48 h, CAT activity and mRNA levels increased by more than 2-fold in heart and skeletal muscle, but not liver or kidney. To examine the effect of insulin deficiency on the M-CPTI (1.1) promoter-CAT reporter gene, we made the mice diabetic by streptozotocin (SZ)-treatment. In the SZ-diabetic transgenic mice, there was a 2 to 3-fold increase in CAT activity and CAT mRNA levels driven by the M-CPTI promoter in heart and skeletal muscle. Insulin treatment of the SZ-diabetic transgenic mice decreased CAT activity and mRNA levels in heart and muscle to that observed with the control insulin sufficient transgenic mice. Feeding a high fat diet for several weeks increased CAT activity and mRNA levels by 2 to 4-fold in heart and skeletal muscle but not liver or kidney of the transgenic mice compared to the control transgenic mice that were fed a low fat diet. Overall, the 1.1-kb 5′-flanking region of the human heart M-CPTI gene that contains the fatty acid response element was shown to be necessary for the tissue-specific hormonal and dietary regulation of the M-CPTI gene expression characteristics similar to those of the endogenous M-CPTI gene. (supported by NIH & AHA).

Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system.

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.

Stimulation of Na,K-ATPase by low potassium requires reactive oxygen species.

The signaling pathway that transduces the stimulatory effect of low K+ on the biosynthesis of Na,K-ATPase remains largely unknown. The present study was undertaken to examine whether reactive oxygen species (ROS) mediated the effect of low K+ in Madin-Darby canine kidney (MDCK) cells. Low K+ increased ROS activity in a time- and dose-dependent manner, and this effect was abrogated by catalase and N-acetylcysteine (NAC). To determine the role of ROS in low-K+-induced gene expression, the cells were first stably transfected with expression constructs in which the reporter gene chloramphenicol acetyl transferase (CAT) was under the control of the avian Na,K-ATPase alpha-subunit 1.9 kb and 900-bp 5'-flanking regions that have a negative regulatory element. Low K+ increased the CAT expression in both constructs. Catalase or NAC inhibited the effect of low K+. To determine whether the increased CAT activity was mediated through releasing the repressive effect or a direct stimulation of the promoter, the cells were transfected with a CAT expression construct directed by a 96-bp promoter fragment that has no negative regulatory element. Low K+ also augmented the CAT activity expressed by this construct. More importantly, both catalase and NAC abolished the effect of low K+. Moreover, catalase and NAC also inhibited low-K+-induced increases in the Na,K-ATPase alpha1- and beta1-subunit protein abundance and ouabain binding sites. The antioxidants had no significant effect on the basal levels of CAT activity, protein abundance, or ouabain binding sites. In conclusion, low K+ enhances the Na,K-ATPase gene expression by a direct stimulation of the promoter activity, and ROS mediate this stimulation and also low-K+-induced increases in the Na,K-ATPase protein contents and cell surface molecules.

Efficient Gene Expression in Skin Wound Sites Following Local Plasmid Injection

Transfection of the skin by local gene delivery, as well as widespread transfection of systemic tissues following intravenous injection of cationic liposome/DNA complexes have been reported. Here, we show that surgically wounded mouse skin can be transfected either by local injection of DNA alone or by intravenous injection of optimized cationic liposome/DNA complexes; however, direct cutaneous injection produces much higher levels of gene expression in the skin, which is targeted to dermal and subdermal layers. High levels of chloramphenicol acetyltransferase activity were present from 3 h to 2 wk following direct injection of a gene expression plasmid into wounded skin and were maintained at detectable levels up to 8 wk after injection. Expression of transferred chloramphenicol acetyltransferase as well as beta-GAL genes was localized to fibroblasts, macrophages, and adipocytes as determined by histochemistry and immunohistochemistry. Further- more, local injection of a human granulocyte- colony-stimulating factor gene expression plasmid produced high levels of the biologically relevant human granulocyte-colony-stimulating factor protein in wounded mouse skin. This efficient and simple method of site-specific gene transfer into wounds may lead to the development of cutaneous gene therapy directed against disorders of abnormal cutaneous wound healing.

NP and L proteins of lymphocytic choriomeningitis virus (LCMV) are sufficient for efficient transcription and replication of LCMV genomic RNA analogs.

The genome of lymphocytic choriomeningitis virus (LCMV) consists of two negative-sense single-stranded RNA segments, designated L and S. Both segments contain two viral genes in an ambisense coding strategy, with the genes being separated by an intergenic region (IGR). We have developed a reverse genetic system that allows the investigation of cis-acting signals and trans-acting factors involved in transcription and replication of LCMV. To this end, we constructed an LCMV S minigenome consisting of a negative-sense copy of the chloramphenicol acetyltransferase (CAT) reporter gene flanked upstream by the S 5' untranslated region (UTR) and IGR and downstream by the S 3' UTR. CAT expression was detected in LCMV-infected cells transfected with the minigenome RNA. Intracellular coexpression of the LCMV minigenome and LCMV L and NP proteins supplied from cotransfected plasmids driven by the T7 RNA polymerase provided by the recombinant vaccinia virus vTF7-3 resulted in high levels of CAT activity and synthesis of subgenomic CAT mRNA and antiminigenome RNA species. Thus, L and NP represent the minimal viral trans-acting factors required for efficient RNA synthesis mediated by LCMV polymerase.

Transient gene expression in mammalian and mosquito cells using a recombinant Semliki Forest virus expressing T7 RNA polymerase.

In this report, we describe a novel recombinant Semliki Forest virus (SFV) expressing T7 RNA polymerase (T7-RP), which was shown to drive transient expression of the chloramphenicol acetyltransferase (cat) gene in mammalian and mosquito cells after transfection of plasmids carrying the reporter gene under the control of the T7 promoter. To our knowledge, this is the first description of a T7-RP-based expression in mosquito cells. Expression of the cat gene was significantly enhanced in mammalian cells by inserting the sequence of the encephalomyocarditis virus internal ribosome entry site between the T7 promoter and the 5' end of the cat gene. In mosquito cells, the level of chloramphenicol acetyltransferase activity was increased by flanking the cat gene with the Autographa californica baculovirus p10 gene 5' and 3' untranslated regions. SFV expressing T7-RP appears, therefore, to be an alternative to other virus-based gene-expression systems in mammalian cells and a powerful tool for protein expression in mosquito cells.

CCAAT Enhancer-binding Protein β and GATA-4 Binding Regions within the Promoter of the Steroidogenic Acute Regulatory Protein (StAR) Gene Are Required for Transcription in Rat Ovarian Cells

Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enhancer-binding protein beta (C/EBPbeta) and a consensus motif for GATA-4 binding (-61/-66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBPbeta were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental -96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBPbeta and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.

Heterologous Gene Expression in Avian Cells: Potential as a Producer of Recombinant Proteins

We have explored the possibility of using avian cells for the expression of human proteins. We found that various avian cells including quail fibrosarcoma cells (QF), duck embryo cells (DE) and primary chicken embryo fibroblasts (CE) could efficiently be transfected with DNA by calcium phosphate coprecipitation, and that promoters which are transcriptionally active in mammalian cells also functioned well in these avian cells. Among the promoters we tested, the major immediate early promoter of human cytomegalovirus drove the highest level of chloramphenicol acetyl transferase (CAT) expression, outperforming the SV40 early promoter and the RSV LTR. Using the bacterial CAT gene as a reporter, we found that levels of CAT activity were higher in QF and DE cells than in mammalian cells such as CHO, HeLa, Vero and 293T cells. We further cloned a sequence encoding human erythropoietin (EPO) and compared its expression in QF and mammalian cells. Consistent with the CAT data, in transient transfection assays, QF cells produced higher levels of EPO than the mammalian cell lines tested. QF cells which can be passaged permanently were stably transfected with an EPO expression vector. The subcloned QF line was able to produce up to 1,700 U/ml EPO from 3 × 10⁶ cells in 72 h. Purified QF-produced EPO showed a broad but discrete protein band, ranging from 33 to 41 kD and was as biologically active as CHO-produced EPO. Although a number of factors still remain to be optimized, our results demonstrate the potential of avian cells such as QF as producers of heterologous proteins.

Estrogen-dependent and independent activation of the P1 promoter of the p53 gene in transiently transfected breast cancer cells.

Loss of p53 function by mutational inactivation is the most common marker of the cancerous phenotype. Previous studies from our laboratory have demonstrated 17 beta estradiol (E2) induction of p53 protein expression in breast cancer cells. Although direct effects of E2 on the expression of p53 gene are not known, the steroid is a potent regulator of c-Myc transcription. In the present studies, we have examined the ability of E2 and antiestrogens to regulate the P1 promoter of the p53 gene which contains a c-Myc responsive element. Estrogen receptor (ER)-positive T47D and MCF-7 cells were transiently transfected with the P1CAT reporter plasmid and levels of CAT activity in response to serum, E2 and antiestrogens were monitored. Factors in serum were noted to be the dominant inducers of chloramphenicol acetyltransferase (CAT) expression in MCF-7 cells. The levels of CAT were drastically reduced when cells were maintained in serum free medium (SFM). However, a subtle ER-mediated induction of CAT expression was detectable when MCF-7 cells, cultured in SFM, were treated with E2. In serum-stimulated T47D cells, the CAT expression was minimal. The full ER antagonist, ICI 182 780 (ICI) had no effect. Treatment with E2 or 4-hydroxy tamoxifen (OHT) resulted in P1CAT induction; OHT was more effective than E2. Consistent with c-Myc regulation of the P1 promoter, E2 stimulated endogenous c-Myc in both cell lines. Two forms of c-Myc were expressed independent of E2 stimuli. The expression of a third more rapidly migrating form was E2-dependent and ER-mediated since it was blocked by the full ER antagonist, ICI, but not by the ER agonist/antagonist OHT. These data demonstrate both ER-mediated and ER-independent regulation of c-Myc and the P1 promoter of the p53 gene, and show differential effects of the two classes of antiestrogens in their ability to induce the P1 promoter of the p53 gene in breast cancer cells.

Fast skeletal muscle-specific expression of a zebrafish myosin light chain 2 gene and characterization of its promoter by direct injection into skeletal muscle.

A zebrafish myosin light chain 2 cDNA clone was isolated and characterized. Sequence analysis of the clone revealed a high homology with the mammalian and avian genes encoding the fast skeletal muscle isoform, MLC2f. In situ hybridization and Northern blot hybridization analyses indicated that the zebrafish MLC2f mRNA is expressed exclusively in the fast skeletal muscle. Ontogenetically, the MLC2f mRNA appears around 16 hours postfertilization (hpf) in the first few well-formed anterior somites. At later stages, the MLC2f mRNA can also be detected in fin buds, eye muscles, and jaw muscles. To develop a useful model system for analyzing muscle gene regulation, the promoter of the zebrafish MLC2f gene was isolated and linked to the chloramphenicol acetyltransferase (CAT) reporter gene. The MLC2f/CAT chimeric constructs were analyzed by direct injection into the zebrafish skeletal muscle, and significant CAT activity was observed; in contrast, little or no CAT activity was generated from a similarly injected prolactin gene promoter/CAT gene construct. Within the 1 kb of the MLC2f promoter region, several MEF2-binding sites and E-boxes were identified, suggesting that MLC2f can be regulated by muscle transcription factors MEF2 and myogenic bHLH proteins. A 5' deletion analysis indicated that the proximal 79 nucleotides from the transcription start site, which contains a single MEF2-binding site, is sufficient to drive a high level of CAT activity in injected muscle. Internal deletion of the MEF2 element in the -79-bp construct caused an 80% decrease in CAT activity, whereas internal deletion of the same MEF2 element in a -1044-bp construct had no effect on induced CAT activity. These observations suggest that an MEF2 element is important to activate the MLC2f gene in muscle cells, and the effect of loss of the proximal MEF2 element can be compensated for by the presence of the upstream MEF2 elements. This study also demonstrated that direct injection of DNA into skeletal muscle is a valid and valuable approach to analyze muscle gene promoters in the zebrafish.

Cell type-specific regulation of CREB gene expression: mutational analysis of CREB promoter activity.

Previous studies have shown that activation of the cyclic AMP (cAMP) pathway down-regulates CREB expression in CATH.a cells, an effect that appears to be mediated via inhibition of CREB gene transcription. In the current study, we compared this effect in CATH.a cells with regulation of CREB expression in another cell line, C6 glioma cells. In contrast to the findings in CATH.a cells, activation of the cAMP pathway up-regulates CREB expression in C6 glioma cells. To determine whether these opposite effects can be explained by regulation of CREB promoter activity, chloramphenicol acetyltransferase (CAT) assays were performed in CATH.a and C6 glioma cells that were transiently transfected with a CREB promoter-CAT fusion plasmid. Activation of the cAMP pathway decreased levels of CAT activity in transfected CATH.a cells but increased CAT activity in transfected C6 glioma cells. We next investigated the effect of mutations in the CREB promoter on such regulation in these two cell lines. Mutations of single CRE or Sp1 binding sites in the CREB promoter reduced basal levels of CAT activity but did not significantly attenuate regulation of the promoter in CATH.a or C6 glioma cells. However, mutation or deletion of two CRE sites in the CREB promoter completely abolished up-regulation of CAT activity in the C6 glioma cells and abolished basal levels of CAT activity in CATH.a cells. CREB promoter activity was also studied in cultured SHSY5Y cells and in primary cultures of striatal neurons as further comparisons. Activation of the cAMP pathway was found to increase CAT activity in both cell types. In the striatal cultures, this effect was obliterated by mutation or deletion of either of the two CREs in the promoter. These findings demonstrate cell type-specific effects of the cAMP pathway on CREB expression, which appear to be mediated via differential regulation of the CREB promoter.

Surfactant Protein B (SP-B) Promoter Function in Transgenic Mice • 309

SP-B, a hydrophobic protein produced by alveolar type II and bronchiolar Clara cells, helps in the spreading and stability of surfactant film and is required for surfactant function. The SP-B gene is regulated both developmentally and hormonally. To study regulatory elements in the SP-B gene promoter in vivo, we made transgenic (tg) mice using a fragment of the human SP-B promoter (-1039/+431bp) linked to the chloramphenicol acetyl transferase (CAT) reporter gene. This SP-B DNA fragment contains both proximal and distal TTF-1 and HNF-3 transcription factor binding sites which promote lung epithelial cell line specificity in vivo. CAT activity in tg lung tissue was significantly increased compared to wild-type (22.2±3.2 vs 0.4±0.1 cpm/mg prot/min; p<0.01), whereas CAT activity in heart, liver, spleen, kidney, intestine, skeletal muscle and brain was not different. In two established tg lines CAT activity was found both in lungs (150 and 264 cpm/mg/min) and in trachea (50 and 125 cpm/mg/min), sites of type II and/or Clara cells in mice. Immunohistochemistry of lung sections using antibodies to CAT and human SP-B showed immunostaining for both proteins localized to type II and Clara cells. In tg fetal lungs, CAT activity was found at 17-19 days gestation and increased ≥4 fold (n=2) during explant culture. In tg adult lung explants (n=3), levels of CAT activity were maintained for at least 3 days (95±13% (se) of preculture). Transforming growth factor β(TGFβ1, 30 ng/ml), a cytokine which downregulates SP-B in human fetal lung explants, reduced CAT activity in both fetal (52±9% of control at 3 days, n=2) and adult mouse lung explants (60±11% of control at 3 days, n=1; 34±3% at 5 days, n=3) whereas35 S-methionine incorporation into total protein was unaffected(101±7%). In contrast, 12-O-tetradecanoyl phorbol-13 acetate (TPA, 30 nM), which also downregulates SP-B in human fetal lung explants, did not reduce CAT activity in tg adult lung ex vivo (182±54%, n=4). We conclude that the human SP-B promoter fragment (-1039/+431bp) exhibits tissue and lung cell specificity in vivo and confers developmental regulation and TGFβ responsiveness ex vivo. These transgenic animals provide a useful in vivo model to study specific regulatory elements in the SP-B promoter.

Transcriptional regulation of the rat poly(ADP-ribose) polymerase gene by Sp1.

Expression of the gene encoding poly(ADP-ribose) polymerase (PARP), although ubiquitous, nevertheless varies substantially between tissues. We have recently shown that Sp1 binds five distinct target sequences (US-1 and F1-F4) in the rat PARP (rPARP) gene promoter. Here we used deletion analyses and site-directed mutagenesis to address the regulatory function played by these Sp1 sites on the basal transcriptional activity directed by the rPARP promoter. Transfection experiments revealed that the most proximal Sp1 site is insufficient by itself to direct any promoter activity. In addition, a weak negative regulatory element was identified between positions -101 and -60. The rPARP promoter directed high levels of chloramphenicol acetyltransferase activity in Jurkat T-lymphoblastoid and Ltk- fibroblast cells but only moderate levels in pituitary GH4C1 and liver HTC cells, correlating with the amounts of PARP detected in these cells by western blot analysis. However, the reduced promoter efficiency in HTC and GH4C1 cells did not result from the lack of Sp1 activity in these cells but suggested that yet uncharacterized regulatory proteins might turn off PARP gene expression by binding negative regulatory elements from the rPARP promoter. Similarly, site-directed mutagenesis on the three most proximal Sp1 elements suggested the influence exerted by Sp1 on the rPARP promoter activity to vary substantially between cell types. It also provided evidence for a basal rPARP promoter activity driven through the recognition of unidentified cis-acting elements by transcription factors other than Sp1.

Increased probability of expression from modified retroviral vectors in embryonal stem cells and embryonal carcinoma cells.

Gene expression from the Moloney murine leukemia retrovirus (Mo-MuLV) is highly restricted in embryonic carcinoma (EC) and embryonic stem (ES) cells. We compared levels of expression in PA317 fibroblasts, F9 (EC) cells, and CCE (ES) cells by Mo-MuLV-based vectors and vectors based on our previously reported MND backbone, which has alterations to address three viral elements implicated as repressors of expression by Mo-MuLV: the enhancer, the primer binding site, and the negative-control region. Expression was evaluated with three reporter genes, the chloramphenicol acetyltransferase (CAT) gene, whose expression was measured by enzymatic assay and by Northern blotting; a truncated nerve growth factor receptor (tNGFR), whose expression was measured by fluorescence-activated cell sorting (FACS) as a cell surface protein; and the enhanced green fluorescent protein (EGFP), whose expression was measured intracellularly by flow cytometry. We found significantly higher levels of CAT activity (5- to 300-fold) and greater quantities of vector-specific transcripts in ES and EC cells transduced with the modified MND-CAT-SN vector than in those transduced with L-CAT-SN. Northern blot analysis indicated that long terminal repeat transcripts from MND-CAT-SN are >80 times more abundant than the L-CAT-SN transcripts. FACS analysis of tNGFR expression from a pair of vectors, L-tNGFR-SN and MND-tNGFR-SN, indicated that only 1.04% of the CCE cells containing the L-tNGFR-SN vector expressed the cell surface reporter, while the MND-tNGFR-SN vector drove expression in 99.54% of the CCE cells. Of the F9 cells containing the L-tNGFR-SN vector, 13.32% expressed tNGFR, while 99.89% of the F9 cells transduced with MND-tNGFR-SN showed expression. Essentially identical results were produced with an analogous pair of vectors encoding EGFP. In unselected pools of F9 cells 48 h posttransduction, the L-EGFP-SN vector drove expression in only 5% of the population while the MND-EGFP-SN vector drove expression in 88% of the cells. After more than 3 weeks in culture without selection, the proportion of cells showing expression from L-EGFP-SN decreased slightly to 3% while expression from the MND-EGFP-SN vector persisted in 80% of the cells. Interestingly, in the few ES and EC cells which did show expression from the L-tNGFR-SN or L-EGFP-SN vectors, the magnitude of reporter expression was similar to that from the MND-tNGFR-SN or MND-EGFP-SN vector in nearly all cells, suggesting that the MND vectors are far less susceptible to position-dependent variegation of expression than are the Mo-MuLV-based vectors. Therefore, the modified retroviral vector, MND, achieves higher net levels of expression due to a greater frequency of expression, which may be useful for the expression of exogenous genes in EC and ES cells.

An extracellular matrix response element in the promoter of the LpS1 genes of the sea urchin Lytechinus pictus.

The extracellular matrix (ECM) has been shown to play an important role in development and tissue-specific gene expression, yet the mechanism by which genes receive signals from the ECM is poorly understood. The aboral ectoderm-specific LpS1-alpha and -beta genes of Lytechinus pictus , members of the Spec gene family, provide an excellent model system to study ECM- mediated gene regulation. Disruption of the ECM by preventing collagen deposition using the lathrytic agent beta-aminopropionitrile (BAPN) inhibits LpS1 gene transcription. LpS1 transcription resumes after removal of BAPN and subsequent collagen reformation. Using a chloramphenicol acetyltransferase (CAT) reporter gene assay, we show that a 125 bp region of the LpS1-beta promoter from -108 to +17 contains an ECM response element (ECM RE). Insertion of the 125 bp region into the promoter of the metallothionein gene of L. pictus, a gene unaffected by ECM disruption, caused the fused promoter to become ECM dependent. As with the endogenous LpS1 genes, CAT activity directed by the fused LpS1-beta promoter resumed in embryos recovered from ECM disruption. A mutation in a cis -acting element called the proximal G-string, which lies in the 125 bp region, caused CAT activity levels in ECM-disrupted embryos to equal that of the wild-type LpS1-bet apromoter in ECM-intact embryos. These results suggest that the intact ECM normally transmits signals to inhibit repressor activity at the proximal G-string in aboral ectoderm cells. Consistent with these results were our findings which showed that in addition to expression in the aboral ectoderm, the proximal G-string mutation caused expression of the CAT gene in oral ectoderm cells. These studies suggested that the proximal G-string serves as a binding site for negative regulation of the LpS1 genes in oral ectoderm during development. We also examined trans -acting factors binding the proximal G-string following ECM disruption. Band shift gels revealed a predominant set of slower migrating nuclear proteins from ECM-disrupted embryos which bound the proximal G-string. This work suggested that ECM disruption initiates signaling that induces a repressor to bind the ECM RE and/or modifies ECM RE binding proteins, which in turn represses LpS1 gene activity.

The role of ATF in regulating the human cytomegalovirus DNA polymerase (UL54) promoter during viral infection.

Previous analysis of the human cytomegalovirus (HCMV) DNA polymerase (UL54) early gene promoter demonstrated that transcriptional activation of this gene is dependent upon the interaction of cellular transcription factors with viral transactivators (J. A. Kerry, M. A. Priddy, T. Y. Jervey, C. P. Kohler, T. L. Staley, C. D. Vanson, T. R. Jones, A. C. Iskenderian, D. G. Anders, and R. M. Stenberg, J. Virol. 70:373-382, 1996). A sequence element, IR1, was shown to be the primary regulatory element of this promoter in transient assays. However, assessment of this element in the context of the viral genome revealed IR1-independent activation at late times after infection. To extend these studies, we aim to identify additional sequence elements involved in the activation of the UL54 promoter. Our present studies demonstrate that the level of binding of proteins to the ATF site in the UL54 promoter is enhanced by viral infection. Furthermore this increase is sensitive to treatment with phosphonoacetic acid (PAA), a DNA synthesis inhibitor. These data suggest that the increase in the level of ATF binding activity is regulated, either directly or indirectly, by HCMV late gene expression. By using specific antibodies, we determined that ATF-1 was a major component of the proteins binding to the UL54 ATF site at late times. In addition, we have demonstrated direct binding of recombinant ATF-1 to the UL54 ATF site. To assess the biological significance of these events, a recombinant virus construct was generated that contained the UL54 promoter with a mutation in the ATF site regulating expression of the chloramphenicol acetyltransferase (CAT) reporter gene inserted between open reading frames US9 and US10. Analysis of this virus (RVATFmCAT) revealed that mutation of the ATF site does not alter the kinetics of UL54 promoter activation. However, levels of CAT mRNA and activity were reduced by 5- to 10-fold compared to those of the wild-type promoter at all stages of infection. These findings indicate that ATF-1 can regulate the levels of UL54 promoter activity at both early and late times. Furthermore, these results imply that HCMV can regulate the activity of cellular factors involved in early gene regulation.

Regulation of the major detoxication functions by phenobarbital and 3-methylcholanthrene in co-cultures of rat hepatocytes and liver epithelial cells.

In the present study, we analysed the expression of monooxygenase activities and mRNAs associated with cytochrome P-450 (CYP), including CYP1A1/2, CYP2B1/2, CYP2C6, CYP2E1, CYP3A1/2, glutathione transferase alpha (GST alpha), aldehyde dehydrogenase and epoxide hydrolase in co-cultures of primary rat hepatocytes and rat liver epithelial cells. We observed that pentoxyresorufin O-deethylation activity was well maintained and ethoxyresorufin O-deethylation activity gradually decreased during co-culture time. In addition, we showed that phenobarbital and 3-methylcholanthrene treatments resulted in a significant increase of these activities. Two general patterns of accumulation of liver-specific mRNAs were observed. CYP1A1/2, CYP2B1/2, CYP3A1/2, GST alpha, aldehyde dehydrogenase and epoxide hydrolase mRNAs were maintained at a stable level, whereas CYP2C6 and CYP2E1 mRNAs showed a continuous decline. In addition, we observed a strong increase of CYP1A1/2 (13.6-fold) and GST alpha (3.9-fold) mRNA expression in 3-methylcholanthrene-treated co-cultures and induction of CYP2B1/2 (19-fold), CYP2C6 (10-fold), CYP3A1/2 (11.2-fold), GST alpha (9-fold), aldehyde dehydrogenase (6-fold) and epoxide hydrolase (5-fold) mRNA expression in phenobarbital-treated co-cultures. Furthermore, we demonstrated that liver-specific gene expression was restricted to hepatocytes, with the notable exception of epoxide hydrolase and CYP2E1 which were expressed in both cell types during the co-culture, as shown by the selective recovery of both hepatocytes and rat liver epithelial cells. Finally, to investigate whether co-cultures could be used to study the molecular mechanisms regulating CYP transcription, we performed transfection of hepatocytes, before the establishment of the co-culture, with large CYP2B1 (3.9 kb) or CYP2B2 (4.5 kb) promoter chloramphenicol acetyltransferase constructs or with a construct containing a 163-bp DNA sequence element reported to confer phenobarbital responsiveness. A 2-3-fold increase over the basal level of chloramphenicol acetyltransferase activity was observed in phenobarbital-treated co-cultures transfected with the phenobarbital-responsive element construct, although phenobarbital had no effect on large CYP2B1 or CYP2B2 promoter fragments. Our results demonstrate that the co-culture system provides a good tool for studying drug metabolism, and shows promise as a new tool for analysing transcriptional regulation under the influence of xenobiotics within primary hepatocytes.

The Pancreatitis-associated Protein I Promoter Allows Targeting to the Pancreas of a Foreign Gene, Whose Expression Is Up-regulated during Pancreatic Inflammation

The pancreatitis-associated protein I (PAP I) is a pancreatic secretory protein expressed in pancreas during acute pancreatitis but not in the healthy pancreas. The promoter of the PAP I gene thus represents a potential candidate to drive expression of therapeutic molecules to the diseased pancreas. In this work, we have constructed recombinant adenoviruses harboring the chloramphenicol acetyltransferase (CAT) gene driven by several fragments of the PAP I promoter and have characterized their properties in vitro and in vivo. In vitro studies showed that the transduction of the pancreatic cell line AR-42J with these adenoviruses led to low levels of CAT activity in basal conditions. After stimulation with a combination of interleukin-6 and dexamethasone or after induction of oxidative stress, CAT activity was strongly induced, a characteristic of the endogenous PAP I gene. Stimulation was maximal when constructs comprised 1253 base pairs of the PAP I promoter, upstream from initiation of transcription, and decreased with shorter fragments of 317, 180, 118 or 61 base pairs. The recombinant adenovirus containing the CAT gene under the control of the PAP I promoter fragment (-1253/+10) was also tested in vivo. Following administration by intravenous injection into mice, CAT activity was measured in several tissues 96 h later. In healthy animals, low but significant CAT activity was detected in pancreas, compared with near background values observed in the other tissues. When experimental acute pancreatitis was induced, CAT expression was strongly enhanced only in pancreas. In control experiments with adenoviruses in which the CAT gene was driven by the cytomegalovirus promoter, higher levels of expression were observed in all tissues. Expression was not modified after induction of acute pancreatitis. In conclusion, this study shows that (i) a recombinant adenovirus containing a fragment of the PAP I promoter allows specific targeting of a reporter gene to the mouse pancreas and (ii) expression of the reporter gene in pancreas is induced during acute pancreatitis. Adenovirus-mediated gene therapy of acute pancreatitis is therefore conceivable.

Repression of the HSV-1 latency-associated transcript (LAT) promoter by the early growth response (EGR) proteins: involvement of a binding site immediately downstream of the TATA box.

During herpes simplex virus (HSV) latency, in neurons of the nervous system, a single family of viral transcripts (the Latency-Associated Transcripts or LATs) are synthesized. Within the LAT promoter region, we have identified a consensus sequence for the EGR proteins in an unusual position immediately downstream of the TATA box. The early growth response (EGR) proteins are rapidly induced in cells by stimuli which also induce HSV to reactivate from latency. In order to determine if EGR proteins play any role in control of LAT transcription, we have analyzed the interactions between EGR proteins and the LAT promoter. Gel retardation and DNase I protection assays demonstrated that EGR1 zinc finger protein bound specifically to the LAT promoter region EGR consensus sequence. To determine if EGR proteins could modulate transcription through the LAT promoter, cotransfection assays were performed using chloramphenicol acetyltransferase (CAT) reporter constructs driven by either the wild-type LAT promoter or a LAT promoter with a mutated EGR binding site. Contransfection of the wild-type LAT promoter construct with EGR expression plasmids resulted in inhibition of the basal level of CAT activity with EGR-2 but not EGR-1 or 3. However, normal levels of CAT activity were observed in cotransfections using the mutant LAT promoter CAT construct suggesting that repression was mediated by the binding of EGR-2 proteins to the LAT promoter. Furthermore, data from combination binding assays using EGR1 and TATA binding protein (TBP) in vitro support the hypothesis that binding of EGR proteins to the LAT promoter prevents binding of TBP and thus suppresses transcription. These results may provide a link between stress responses in neurons of the CNS which activate the EGR family of proteins and HSV reactivation from latency due to the same stress response.

The human gene for the regulatory subunit RI alpha of cyclic adenosine 3', 5'-monophosphate-dependent protein kinase: two distinct promoters provide differential regulation of alternately spliced messenger ribonucleic acids.

The present study reports the exon-intron organization of the human RI alpha gene of cAMP-dependent protein kinase and approximately kilobases (kb) of the 5'-flanking region obtained by isolation and sequencing of several phage clones from human genomic libraries. The RI alpha gene is composed of nine coding exons of varying lengths, separated by introns, giving the gene a total length of at least 21 kb. our recent cloning of a processed RI alpha pseudogene with a 5'-noncoding region different from the previously reported RI alpha complementary RNA indicated that the RI alpha gene may have multiple leader exons giving rise to alternately spliced messenger RNAs (mRNAs). Reverse transcription of human testis RNA followed by PCR identified two different RI alpha mRNA species (RI alpha 1a and RI alpha 1b) containing distinct sequences due to alternately splicing the gene. The previously known RI alpha 1b mRNA revealed low constitutive expression in a human B lymphoid cell line (Reh) and was stimulated only 4- to 6-fold by treatment with cAMP. In contrast, very low levels of the novel RI alpha 1a mRNA were present in untreated Reh cells, but were stimulated 40-to 50-fold by cAMP. The 5'-flanking sequence of the RI alpha gene was G/C rich and did not contain any TATA box. Several putative transcription initiation sites were identified in front of each leader exon (exons 1a and 1b) by the 5'-rapid amplification of complementary DNA ends technique. To determine whether the sequences 5' of both leader exons had promoter activities, the 5'-flanking sequences of exons 1a and 1b were inserted in front of a chloramphenicol acetyltransferase reporter gene, and their ability to direct transcription were examined. Transfection of these constructs into rat GH4C1 cells demonstrated that both constructs had promoter activities, as evidenced by high levels of chloramphenicol acetyltransferase activity.