Expression Of Chloramphenicol Acetyltransferase Gene Research Articles

RNA Chaperone Function of a Universal Stress Protein in Arabidopsis Confers Enhanced Cold Stress Tolerance in Plants.

The physiological function of Arabidopsis thaliana universal stress protein (AtUSP) in plant has remained unclear. Thus, we report here the functional role of the Arabidopsis universal stress protein, AtUSP (At3g53990). To determine how AtUSP affects physiological responses towards cold stress, AtUSP overexpression (AtUSP OE) and T-DNA insertion knock-out (atusp, SALK_146059) mutant lines were used. The results indicated that AtUSP OE enhanced plant tolerance to cold stress, whereas atusp did not. AtUSP is localized in the nucleus and cytoplasm, and cold stress significantly affects RNA metabolism such as by misfolding and secondary structure changes of RNA. Therefore, we investigated the relationship of AtUSP with RNA metabolism. We found that AtUSP can bind nucleic acids, including single- and double-stranded DNA and luciferase mRNA. AtUSP also displayed strong nucleic acid-melting activity. We expressed AtUSP in RL211 Escherichia coli, which contains a hairpin-loop RNA structure upstream of chloramphenicol acetyltransferase (CAT), and observed that AtUSP exhibited anti-termination activity that enabled CAT gene expression. AtUSP expression in the cold-sensitive Escherichia coli (E. coli) mutant BX04 complemented the cold sensitivity of the mutant cells. As these properties are typical characteristics of RNA chaperones, we conclude that AtUSP functions as a RNA chaperone under cold-shock conditions. Thus, the enhanced tolerance of AtUSP OE lines to cold stress is mediated by the RNA chaperone function of AtUSP.

Regulating effects of insertion direction of matrix attachment regions on transgenic expression in stably transformed Chinese hamster ovary cells.

We investigated the effects of different directions of insertion of matrix attachment region (MAR) sequences on transgenic expression in stably transformed Chinese hamster ovary (CHO) cells. The MAR sequences were inserted in forward or reverse directions into the expression vectors, and transfected into CHO cells. The expression of the chloramphenicol acetyltransferase (CAT) reporter gene and the relative copy numbers of the CAT gene were analyzed. The CAT gene expression levels in the vector with the MAR sequence inserted in the forward or reverse directions increased compared with expression without the MAR sequence. The relative copy numbers of the CAT gene with MAR sequenced vectors inserted in the reverse and forward directions were lower, than in the control group. The direction of insertion of MAR sequences had no significant effect on expression levels. The expression levels were not proportional to the copy numbers of the gene.

Positional effects of the matrix attachment region on transgene expression in stably transfected CHO cells

Previous work has shown that the MAR (matrix attachment region) could increase transgene expression in stably transfected CHO (Chinese-hamster ovary) cells. To study the positional effect of MAR on transgene expression, three expression vectors were constructed which contained the human beta-globin MAR in different sites, including the vector with two MARs flanking the CAT (chloramphenicol acetyltransferase) expression cassette, one MAR at the 59 or 39 site. These vectors were transfected into CHO cells. The level of CAT gene expression was most effectively increased by two MARs flanking the CAT expression cassette. This increase was also seen when MAR was inserted at the 59 site upstream of the expression cassette, whereas the transgene expression level decreased when MAR was inserted at the 39 site downstream of the expression cassette. We have also shown that the transgene expression level is not directly proportional to the gene copy number, and gene copy number dependency does not exist.

Identification of a Novel Transcriptional Repressor (HEPIS) That Interacts with nsp-10 of SARS Coronavirus

A novel gene was previously isolated from a cDNA library of human embryo lung tissue by its encoded protein, which interacts with non-structural protein 10 (nsp-10) of the severe acute respiratory syndrome coronavirus (SARS-CoV). The protein was named human embryo lung cellular protein interacting with SARS-CoV nsp-10 (HEPIS), and it is composed of 147 amino acids with several CK II phosphorylation sites. In the present study, we demonstrated that HEPIS was capable of suppressing chloramphenicol acetyltransferase (CAT) gene expression controlled by different enhancerelements in a transcription assay. HEPIS interacted specifically with the HSP70 TATA sequence, and not with various other enhancer elements in a binding test. Furthermore, we co-immunoprecipitated HEPIS with BTF3, a component of the RNA pol II initiation complex, and observed reduced proliferation of HeLa cells transfected with the HEPIS gene. Taken together, our results suggest that HEPIS may function as a potential transcriptional repressor.

Increasing transient expression of CAT gene in Porphyra haitanensis by Matrix attachment regions and 18S rDNA targeted homologous recombination

To test whether matrix attachment regions (MARs) and 18S rDNA can in£uence CAT gene transient expression positively in the red algae Porphyra haitanensis, a targeting vector pHR-CAT containing a portion of the 18S rDNA from P. haitanensis ,p MAR1-HR-CAT containing one MAR from silkworm and a portion of the 18S rDNA from P. haitanensis and pMAR2-HRCAT containing two MARs from silkworm and a portion of the 18S rDNA from P. haitanensis were constructed. With the electroporation method, the vectors were transferred into the protoplasts from the thalli of P. haitanensis. The results showed that the expression of chloramphenicol acetyl transferase (CAT) protein in transformed cells reached a maximum at 96 h after transformation. It was increased markedly with the pMAR2-HR-CAT compared with the pHR-CAT or the pCAT @ 3-control vector (Po0.01), and it was increased inconspicuously with pHR-CAT compared with the pCAT @ 3-control vector (P40.05). It is suggested that MAR from silkworm could enhance the transient expression of foreign genes in P. haitanensis.

Repression of 5-aminolevulinate synthase gene by the potent tumor promoter, TPA, involves multiple signal transduction pathways

The potent tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA) induces activator protein-1 (AP-1) transcription factors, early response genes involved in a diverse set of transcriptional regulatory processes, and protein kinase C (PKC) activity. This work was designed to explore the signal transduction pathways involved in TPA regulation of 5-aminolevulinate synthase (ALAS) gene expression, the mitochondrial matrix enzyme that catalyzes the first and rate-limiting step of heme biosynthesis. We have previously reported that TPA causes repression of ALAS gene, but the signaling pathways mediating this effect remain elusive. The present study investigates the role of different cascades often implicated in the propagation of phorbol ester signaling. To explore this, we combined the transient overexpression of regulatory proteins involved in these pathways and the use of small cell permeant inhibitors in human hepatoma HepG2 cells. In these experimental conditions, we analyzed TPA action upon endogenous ALAS mRNA levels, as well as the promoter activity of a fusion reporter construct, harboring the TPA-responsive region of ALAS gene driving chloramphenicol acetyl transferase gene expression. We demonstrated that the participation of α isoform of PKC, phosphatidylinositol 3-kinase (PI3K), extracellular-signal regulated kinase (ERK1/2), and c-Jun N-terminal kinase (JNK) is crucial for the end point response. Remarkably, in this case, ERK activation is achieved in a Ras/Raf/MEK-independent manner. We also propose that p90 RSK would be a convergent point between PI3K and ERK pathways. Furthermore, we elucidated the crosstalk among the components of the cascades taking part in TPA-mediated ALAS repression. Finally, by overexpression of a constitutively active p90 RSK and the coactivator, cAMP-response element protein (CREB)-binding protein (CBP), we reinforced our previous model, that implies competition between AP-1 and CREB for CBP.

Effect of 3' terminal codon pairs with different frequency of occurrence on the expression of cat gene in Escherichia coli.

In a previous study, we have identified four types of 3' terminal codon pairs depending on their frequency of occurrence in the Escherichia coli genome: overrepresented, moderately represented, underrepresented, and missing. In this study, the influence of eight codon pairs belonging to these four groups on the efficiency of chloramphenicol acetyltransferase ( cat) gene expression in E. coli is examined. Our results show that the missing codon pairs CCU:UAG (Pro:Stop) and CCC:UAG (Pro:Stop) had decreasing effect, whereas another missing pair CCU:AGG (Pro:Arg) had an opposite effect on the yield of CAT protein in comparison with the wild-type cat gene.

Agonist-Activated Glucocorticoid Receptor Inhibits Binding of Heat Shock Factor 1 to the Heat Shock Protein 70 Promoterin Vivo

We have previously shown that activation of glucocorticoid receptor (GR) signaling in stressed cells will cause inhibition of the heat shock response as mediated by heat shock transcription factor 1 (HSF1). In that work, a full-length human heat shock protein 70 (Hsp70) promoter was used to measure HSF1 transactivity, and the data suggested inhibition of HSF1 through the transactivation or transrepressive properties of GR. Here, we show that the inhibitory effect of glucocorticoid agonist (dexamethasone) upon Hsp70 promoter activity is rapid, occurring within 1 h of hormone addition. Moreover, addition of hormone during the first hour of recovery from stress was sufficient to inhibit HSF1. Thus, dexamethasone is able to rapidly reverse HSF1 transactivity, suggesting a transrepressive mode of action for GR. Yet, GR transrepression of HSF1 by analysis of putative negative glucocorticoid response elements in the Hsp70 promoter was not found. To further investigate the in vivo nature of this fast-acting mechanism, we used the chromatin immunoprecipitation assay with primers specific to the human Hsp70 promoter. Dexamethasone inhibited HSF1 binding at the Hsp70 promoter in response to heat or chemical shock (sodium arsenite). Moreover, dexamethasone also blocked promoter binding by a constitutively active mutant of HSF1 (hHSF1-E189) expressed under nonstress conditions. In all cases, inhibition of HSF1 recruitment to the promoter by dexamethasone was blocked by the GR antagonist RU486, a result that was consistent with promoter activity based on chloramphenicol acetyl transferase gene expression. The ability of dexamethasone to prevent HSF1 recruitment to the promoter was fast acting (occurring in as little as 15 min), and the hormone also caused release of HSF1 already bound to the promoter. Although these results suggest GR can effectively prevent HSF1 binding to Hsp promoters, fractionation and Western blot experiments showed that stress-activated HSF1 was not released from the nucleus in response to hormone. Thus, this effect of dexamethasone is either specific to the Hsp70 promoter or causes shunting of HSF1 to other high-affinity nuclear sites. These observations provide evidence of a novel mechanism for attenuation of the heat shock response by glucocorticoids: prevention or reversal of HSF1 recruitment to Hsp promoters through the rapid actions of GR.

Mouse muscle identity: The position‐dependent and fast fiber‐specific expression of a transgene in limb muscles is methylation‐independent and cell‐autonomous

We previously characterised transgenic mice in which fast-muscle-specific regulatory sequences from the human aldolase A pM promoter drive the chloramphenicol acetyltransferase gene expression. Mutation of a NF1/MEF2 binding site (M2 motif) in this promoter does not affect fibre-type specificity of the transgene but modifies its expression in a subset of fast-twitch fibres at the limb level, preferentially affecting distal limb muscles. We investigated the molecular and cellular bases of this peculiar expression pattern that provided an adequate model to characterise the mechanisms responsible for muscle positional information. By direct electrotransfer of mutated M2 construct in adult muscle, we demonstrate that positional differences in mutated M2 transgene expression are not observed when the transgene is not integrated into chromatin. Also, this transgene expression pattern does not seem to be correlated with the extent of CpG methylation in its promoter sequence. Finally, we show that positional values reflected by CAT levels are maintained in primary cultures established from different adult limb muscles, as well as in heterotopically transplanted muscles. Our results suggest that mutation of the M2 site contributes to reveal a molecular memory of fibre fate that would be set up on pM promoter during development and persist into adulthood possibly through a chromatin imprint maintained in satellite cells associated with various limb muscles.

Tetracycline-inducible gene expression in Trichomonas vaginalis.

Transient and stable gene delivery systems are available for Trichomonas vaginalis, however, they do not allow regulated expression of target genes. To study essential genes or proteins that are toxic to the cells when over expressed, we have developed an inducible/repressible gene expression system in this parasite, which is driven by the tet-operator (tetO) and regulated tetracycline-responsive Tet repressor (TetR). Inducible chloramphenicol acetyl transferase (CAT) gene expression is observed using a concentration of tetracycline (Tc) as low as 0.1 microg x ml(-1). Expression increases with drug dose with a maximum level of CAT induction achieved in stable transfectants using 5 microg x ml(-1) Tc. CAT protein expression is detectable within 12 h and reaches a maximum level at 48 h, demonstrating that inducible expression is time and dose-dependent. In an inverse experiment, parasites previously cultivated with 1 microg x ml(-1) of Tc for 48 h, were grown in the absence of drug to determine the kinetics of repression. A significant decrease in protein concentration is detected after 48 h, and no detectable protein is observed after 72 h. Experiments replacing the CAT gene with the puromycin N-acetyltransferase (PAC) gene in the Tet regulated expression construct have demonstrated the use of this system for testing putative toxic and essential genes. The establishment of regulated gene expression of exogenous genes in T. vaginalis represents a crucial step towards determining the function of proteins in this divergent parasite.

The 5′-Flanking Region of the Murine Epididymal Protein of 17 Kilodaltons Gene Targets Transgene Expression in the Epididymis

A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5'-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5'-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.

Analysis of regulatory functions for the region located upstream from the latency-associated transcript (LAT) promoter of pseudorabies virus in cultured cells

The latency-associated transcript (LAT) promoter of pseudorabies virus (PrV) is unique among the many promoters of the viral genome in that it remains active during the latent state. The regulatory mechanism of PrV LAT gene expression is complex and different between latency and lytic infection of cultured cells. Although two different sequences, LAP1 and LAP2, are thought to be involved in LAT gene expression, the function of the upstream region of the LAT promoter (LAP1 and LAP2) remains an enigma, even in cultured cells. To analyze the function of the upstream region, it is necessary to examine the effects of the upstream sequence on LAT gene expression in the absence of other viral proteins. Transient expression assays were performed by employing a series of reporter plasmids in which various sequences upstream of the LAT promoter (from nucleotide positions −592 to +423 relative to the transcriptional start site of the large latency transcript (LLT)) were linked to the chloramphenicol acetyltransferase (CAT) gene in cells of neuronal and non-neuronal origin. We identified a region (from nucleotide positions −3606 to −1386) that was capable of repressing the LAT promoter activity in Vero cells by analyzing CAT gene expression of the series of reporter plasmids. This effect was not observed in Neuro-2a cells. We have also shown that the LAT promoter activity of the reporter plasmid containing the upstream region was repressed by the immediate-early gene product IE180 in Vero cells, but not in Neuro-2a cells. These results suggest that the upstream region of the LAT promoter may have a role in repressing LAT gene expression in cultured non-neuronal cells.

A TEF-1-element is required for activation of the promoter of pseudorabies virus glycoprotein X gene by IE180.

The pseudorabies virus (PRV) immediate-early regulatory protein IE180 is able to transactivate the viral early and late genes. Using chloramphenicol acetyltransferase (CAT) assay, we investigated the transactivation function of IE180 to the promoter of PRV glycoprotein X (gX) gene, and our results showed that IE180 could significantly increase the expression of CAT gene which was under the control of gX promoter. To further identify the activation domains of IE180 protein that interact with the gX promoter sequences, various truncated mutants of IE180 gene and gX promoter gene were constructed and analyzed by CAT and gel retardation assay. Results revealed that the N-terminal amino acid residues from 133 to 736 of IE180 could interact with the binding site of transcriptional enhancer factor-1 (TEF-1) that resides in the gX promoter. Formation of protein-DNA complexes between the IE180 protein and the TEF-1 element of the gX promoter was observed using electrophoretic mobility shift assay (EMSA) as well as Southwestern blot analysis. These results indicated that a direct interaction occurred between IE180 and the TEF-1 element; and this interaction was abolished if the TEF-1 element was mutated. The association of IE180 with the TEF-1 element was further confirmed by the supershift of EMSA complexes using IE180 specific antibody. Taken together, our results suggested that formation of a complex between the IE180 protein and TEF-1 element in the gX promoter region was involved in the transcriptional regulation of the gX gene.

Substance P enhances HIV-1 replication in latently infected human immune cells

Substance P (SP) is a potent modulator of neuroimmunoregulation. SP receptors are present on human monocytes and T lymphocytes, and SP alters the function of these immune cells. We investigated the effects of SP on HIV-1 replication in latently infected human immune cells. SP significantly enhanced HIV-1 replication in the latently infected promonocytic cell line (U1) and T lymphocyte line (ACH-2) stimulated with tumor necrosis factor (TNF-α). When added to these cells in combination with TNF-α, SP also enhanced HIV-1 gag gene expression in U1 and ACH-2 cells. This stimulatory effect of SP was associated with the activation of HIV-LTR (long terminal repeat) driven chloramphenicol acetyltransferase (CAT) gene expression, and could be blocked by pretreatment of U1 and ACH-2 cells with an SP receptor antagonist RP-67,580, indicating specific SP receptor-mediated regulation. Furthermore, the addition of SP to the cultures of latently infected peripheral blood mononuclear cells isolated from HIV-1-infected patients enhanced HIV-1 gag gene expression. Thus, SP may play a potentially important role as a positive regulator of HIV-1 replication in latently infected monocytes and lymphocytes. These observations may have significant implications toward understanding the role of neuropeptide SP in the immunopathogenesis of HIV-1 infection and AIDS.

Inhibition of Heat Shock Transcription Factor by GR

The GR is a hormone-activated transcription factor that acts to regulate specific gene expression. In the absence of hormone, the GR and other steroid receptors have been shown to form complexes with several mammalian heat shock proteins. As heat shock proteins are produced by cells as an adaptive response to stress, speculation has existed that communication between the heat shock and glucocorticoid hormone signal pathways must exist. Only recently has evidence to support this hypothesis been reported. In almost all cases, the evidence has been of an ability of heat shock to cause a potentiation of the glucocorticoid hormone response. In this proposal, evidence is now presented that heat shock signaling can, in turn, be regulated by glucocorticoids. In mouse L929 cells stably expressing a chloramphenicol acetyltransferase reporter controlled by the human heat shock protein70 promoter and containing known binding sites for heat shock transcription factor 1 treatment with glucocorticoid agonist (dexamethasone) results in a dose-dependent decrease of stress-induced chloramphenicol acetyltransferase gene expression. In these cells, inhibition of heat shock protein70 promoter activity by dexamethasone was completely blocked by GR antagonist (RU486). Similar treatment of L929 cells stably expressing a chloramphenicol acetyltransferase reporter under the control of the constitutively active SV40 promoter showed no such inhibition by dexamethasone. More importantly, dexamethasone was also found to inhibit heat shock-induced expression of the major heat shock proteins-heat shock proteins70, 90, and 110. Thus, the inhibitory effect of dexamethasone appears to apply to most, if not all, heat shock transcription factor 1-regulated genes. Although dexamethasone did not prevent the DNA-binding function of heat shock-activated heat shock transcription factor 1, it did inhibit a constitutively active mutant of human heat shock transcription factor 1 under nonstress conditions, suggesting that dexamethasone repression of heat shock transcription factor 1 was primarily through an inhibition of heat shock transcription factor 1 transcription enhancement activity. To more accurately characterize the stage of GR signaling responsible for inhibition of heat shock transcription factor 1, a series of Chinese hamster ovary cells containing either no GR, wild-type mouse GR, or single-point mutations of GR were employed. Dexamethasone inhibition of heat shock-induced heat shock transcription factor 1 activity was observed in the presence of wild-type GR, but not in Chinese hamster ovary cells lacking GR, suggesting that signaling cascades other than GR were not involved in this effect of dexamethasone. Consistent with this conclusion was the observation that dexamethasone had no effect on activity of the MAPKs (ERK1, ERK2, or c-jun N-terminal kinase), which are known to negatively regulate heat shock transcription factor 1. Dexamethasone inhibition of heat shock transcription factor 1 was not seen in Chinese hamster ovary cells expressing GR defective for DNA-binding function. Moreover, dissociation of GR/Hsp90/Hsp70 complexes was observed in response to hormone for both the wild-type and DNA binding-defective forms of GR, demonstrating that release of Hsp90 or Hsp70 (both of which are known to keep heat shock transcription factor 1 in its inactive state) could be ruled out as a potential mechanism. Thus, it appears that GR-mediated transactivation or transrepression is required for the inhibitory effect of dexamethasone on heat shock transcription factor 1 activity. Taken as a whole, these results provide evidence for a novel mechanism of cross-talk in which signaling by the GR can attenuate the heat shock response in cells through an inhibition of the transcription enhancement activity of HSF1.

The 3′‐untranslated region of human interleukin‐8 mRNA suppresses IL‐8 gene expression

Although adenosine/uridine (AU)-rich sequences in the 3'-untranslated region (UTR) of the interleukin-8 (IL-8) gene have been suggested to contribute to its post-transcriptional regulation, the molecular basis whereby this occurs still needs to be understood. To investigate the role of the 3'-UTR on human IL-8 gene regulation, chimeric reporter genes were generated by adding full length or differentially deleted 3'-UTR of the IL-8 gene to chloramphenicol acetyltransferase (CAT). Addition of the entire IL-8 3'-UTR markedly reduced CAT mRNA and protein expression in COS 7 cells. In a reporter gene study, IL-8 3'-UTR destabilized CAT mRNA, which was dependent on active transcription in COS 7 cells. A 357-base sequence (nucleotides (nt) 2387-2743 of genomic DNA) within 3'-UTR, designated e, suppressed CAT gene expression by accelerating CAT mRNA turnover. A 26-base AU-rich sequence (nt 2552-2577) within e, containing four AUUUA pentamers that form two UAUUUAUU and one UUAUUUAU octamers, did not suppress CAT gene expression. However, deletion of the AU-rich sequences attenuated the inhibitory effect of e on CAT gene expression. Elimination of the first 100 bases (nt 2386-2486) attenuated the potency of fragment e, but much weaker than elimination of the first 146 bases (nt 2387-2533). This study gives new insights in unravelling the molecular mechanisms involved in the post-transcriptional regulation of the IL-8 gene.

In Vivo Gene Electroporation Confers Strong Transient Expression of Foreign Genes in the Chicken Testis

To attain foreign gene expression in vivo in the testis of living chickens, chloramphenicol acetyltransferase (CAT) and firefly luciferase reporter genes were transfected by electroporation (EP). Bioluminescence imaging indicated clear expression of the luciferase reporter gene localized in and around the injection site of the chicken testis. The CAT activity decreased sharply from 7 to 14 d posttransfection (P < 0.01) and remained low until 28 d. The presence of the self-replication sequence of Epstein-Barr virus did not give significantly higher CAT gene expression over the 28-d posttransfection. The results suggest that in vivo gene EP confers strong, likely transient, foreign gene expression in the testis of living chickens.

The efficiency of different IRESs (internal ribosomes entry site) in monocistronic mRNAS.

The IRES from poliovirus and from encephalomyocarditis virus (EMCV) added between the cap and the AUG initiator codon were strong inhibitors of chloramphenicol acetyltransferase gene expression in three different cell types. The poliovirus IRES also inhibited bGH (bovine growth hormone) cDNA expression in the HC11 mammary cell line when added between the rabbit whey acidic gene promoter and the cDNA whereas the HTLV-1 IRES showed a stimulatory effect in the same situation. RNA stem loops were added before HTLV-1 (SUR) and the BiP (Immunoglobulin heavy-chain Binding Protein) IRESs followed by the firefly luciferase gene under the control of Rous sarcoma virus (RSV) promoter. The RNA loops abolished the expression of the reporter gene almost completely. These data suggest that the different IRESs may favour or inhibit translation of monocistronic mRNA.

Vitamin D receptor interactions with the rat parathyroid hormone gene: synergistic effects between two negative vitamin D response elements.

Vitamin D response elements (VDREs) that are required for negative regulation of rat parathyroid hormone (rPTH) gene expression have been characterized. Gel mobility shift assays using DNA restriction enzyme fragments and recombinant proteins for vitamin D and retinoic acid X receptors (VDR/RXR) revealed a sequence between -793 and -779 that bound a VDR/RXR heterodimer with high affinity (VDRE1). Furthermore, a lower affinity site (VDRE2) was detected that acted in combination with VDRE1 to bind a second VDR/RXR complex. As determined by ethylation interference analysis, the nucleotide sequence of VDRE1 consisted of GGTTCA GTG AGGTAC, which is remarkably similar to the sequence of the negative VDRE found in the chicken PTH (cPTH) gene. Using the same technique, VDRE2 was identified between positions -760 and -746 and contained the sequence AGGCTA GCC AGTTCA. Functional analysis was determined by transfection studies with plasmid constructs that expressed the gene for chloramphenicol acetyl transferase (CAT). The ability of the VDREs to regulate gene expression was tested in their native context with the rPTH promoter as well as when positioned immediately upstream from the cPTH promoter. With either plasmid construct, exposure to 10(-8)M 1,25(OH)2D3 resulted in a 60-70% decrease in CAT gene expression when both VDRE1 and VDRE2 were present. Examination of the individual VDREs showed that inhibition by 10(-8) M 1,25(OH)2D3 was only 35-40% when just VDRE1 was present. By itself, VDRE2 was even less effective, as significant inhibition of CAT activity (20%) was observed only in the presence of higher concentrations of 1, 25(OH)2D3 (10(-7)M) or when a plasmid vector that overexpressed the VDR protein was cotransfected. In conclusion, the rPTH gene contains two negative VDREs that act in concert to bind two RXR/VDR heterodimer complexes and that both VDREs are required for maximal inhibition by 1,25(OH)2D3.

Genetic targeting: the serotonin N-acetyltransferase promoter imparts circadian expression selectively in the pineal gland and retina of transgenic rats.

The arylalkylamine N-acetyltransferase (AA-NAT) gene is strongly expressed in the rat primarily in the pineal gland; low levels of expression are also found in the retina. AA-NAT catalyzes the key regulatory step controlling rhythmic melatonin output: the acetylation of serotonin. In the rat, the AA-NAT gene is expressed at night. This is controlled partly by cyclic AMP (cAMP) acting through a composite cAMP-responsive element-CCAAT site located upstream of the transcription start point. In the present study, we have extended our previous in vitro findings and found that additional elements in the 5' flanking region and first intron play an important role in the regulation of the AA-NAT gene. This led us to test the influence of an AA-NAT 5' flanking segment on the expression of the bacterial chloramphenicol acetyltransferase gene in a rat transgenic model. The results of this study clearly demonstrate that the segment of the AA-NAT gene that encompasses the minimal promoter and the first intron is able to confer the highly specific pineal/retinal and time-of-day patterns of AA-NAT gene expression. This advance also provides a tool that selectively targets genetic expression to pinealocytes and retinal photoreceptors, providing new experimental opportunities to probe gene expression in these tissues.