Transcription Of Chloramphenicol Acetyltransferase Research Articles

Transformation of diatom Phaeodactylum tricornutum by electroporation and establishment of inducible selection marker.

Diatoms are important primary producers in the marine ecosystem. Currently it is difficult to genetically transform diatoms due to the technical limitations of existing methods. The promoter/terminator of the nitrate reductase gene of the model diatom Phaeodactylum tricornutum was cloned and used to drive chloramphenicol acetyltransferase (CAT) reporter gene expression. The construct was transferred by electroporation into P. tricornutum grown in medium lacking silicon. CAT expression was induced in transformed diatoms in the presence of nitrate, enabling growth in selective medium, and was repressed when ammonium was the only nitrogen source. Expression of CAT transcript and protein were demonstrated by RT-PCR and Western blot analysis, respectively. Our study is the first to report a successful genetic transformation of diatom by electroporation in an economical and efficient manner and provides a tightly regulated inducible gene expression system for diatom.

Acute Inhibition of Fatty Acid Import Inhibits GLUT4 Transcription in Adipose Tissue, but Not Skeletal or Cardiac Muscle Tissue, Partly Through Liver X Receptor (LXR) Signaling

OBJECTIVEInsulin-mediated glucose uptake is highly sensitive to the levels of the facilitative GLUT protein GLUT4. Transcription of the GLUT4 gene is repressed in states of insulin deficiency and insulin resistance and can be induced by states of enhanced energy output, such as exercise. The cellular signals that regulate GLUT4 transcription are not well understood. We hypothesized that changes in energy substrate flux regulate GLUT4 transcription.RESEARCH DESIGN AND METHODSTo test this hypothesis, we used transgenic mice in which expression of the chloramphenicol acetyltransferase (CAT) gene is driven by a functional 895-bp fragment of the human GLUT4 promoter, thereby acting as a reporter for transcriptional activity. Mice were treated with a single dose of etomoxir, which inhibits the transport of long-chain fatty acids into mitochondria and increases basal, but not insulin-mediated, glucose flux. GLUT4 and transgenic CAT mRNA were measured.RESULTSEtomoxir treatment significantly reduced CAT and GLUT4 mRNA transcription in adipose tissue, but did not change transcription in heart and skeletal muscle. Downregulation of GLUT4 transcription was cell autonomous, since etomoxir treatment of 3T3-L1 adipocytes resulted in a similar downregulation of GLUT4 mRNA. GLUT4 transcriptional downregulation required the putative liver X receptor (LXR) binding site in the human GLUT4 gene promoter in adipose tissue and 3T3-L1 adipocytes. Treatment of 3T3-L1 adipocytes with the LXR agonist, TO901317, partially restored GLUT4 expression in etomoxir-treated cells.CONCLUSIONSOur data suggest that long-chain fatty acid import into mitochondria in adipose tissue may produce ligands that regulate expression of metabolic genes.

Reactive Oxygen Species Regulate Ceruloplasmin by a Novel mRNA Decay Mechanism Involving Its 3′-Untranslated Region: IMPLICATIONS IN NEURODEGENERATIVE DISEASES

Ceruloplasmin (Cp), a copper-containing protein, plays a significant role in body iron homeostasis as aceruloplasminemia patients and Cp knock-out mice exhibit iron overload in several tissues including liver and brain. Several other functions as oxidant, as antioxidant, and in nitric oxide metabolism are also attributed to Cp. Despite its role in iron oxidation and other biological oxidation reactions the regulation of Cp by reactive oxygen species (ROS) remains unexplored. Cp is synthesized in liver as a secretory protein and predominantly as a glycosylphosphatidylinositol-anchored membrane-bound form in astroglia. In this study we demonstrated that Cp expression is decreased by an mRNA decay mechanism in response to extracellular (H2O2) or intracellular oxidative stress (by mitochondrial chain blockers rotenone or antimycin A) in both hepatic and astroglial cells. The promotion of Cp mRNA decay is conferred by its 3'-untranslated region (UTR). When chloramphenicol acetyltransferase (CAT) gene was transfected as a chimera with Cp 3'-UTR in hepatic or astroglial cells, in response to either H2O2, rotenone, or antimycin A, the expression of CAT transcript was decreased, whereas expression of a 3'-UTR-less CAT transcript remained unaffected. RNA gel shift assay showed significant reduction in 3'-UTR-binding protein complex by ROS in both cell types that was reversed by the antioxidant N-acetylcysteine suggesting that ROS affects RNA-protein complex formation to promote Cp mRNA decay. Our finding is not only the first demonstration of regulation of Cp by ROS by a novel post-transcriptional mechanism but also provides a mechanism of iron deposition in neurodegenerative diseases.

Translation Toeprinting Assays Using Fluorescently Labeled Primers and Capillary Electrophoresis

The protocol described here is an adapted version of the toeprinting assay in which the oligonucleotide used to prime the reverse transcription step is labeled with a fluorescent dye instead of 32P. By using a fluorescent dye, the results of the assay are obtained within one hour by direct electrophoresis of the samples on an automated sequencing machine. This eliminates the need to cast and run polyacrylamide gels and to wait to expose dried gels. We show that an identical toeprint was found for the chloramphenicol acetyltransferase transcript using this nonradioactive method, which is in agreement with the previously published 32P-labeled method. Furthermore, in addition to being a faster and safer method, a larger region of sequence can be analyzed with one primer in a single experiment.

Effects of 3′ Terminus Modifications on mRNA Functional Decay during in Vitro Protein Synthesis

The pcnB gene, which encodes the principal poly(A) polymerase of Escherichia coli, promotes 3'-polyadenylation and chemical decay of mRNA. However, there is no evidence that pcnB-mediated mRNA destabilization decreases protein synthesis, suggesting that polyadenylation may enhance translational efficiency. Using in vitro translation by E. coli cell extracts and toeprinting analysis of transcripts encoded by the chloramphenicol acetyltransferase (CAT) and beta-galactosidase genes to investigate this notion, we found no effect of poly(A) tails on protein synthesis. However, we observed that 3'-polyguanylation delayed the chemical decay of CAT mRNA and, even more dramatically, increased the ability of CAT mRNA to produce enzymatically active full-length protein in 30 S E. coli cell fractions. This resulted from interference with the primary mechanism for inactivation of CAT transcript function in cell extracts, which occurred by 3'-exonucleolytic degradation rather than endonucleolytic fragmentation by RNase E. Using bacteriophage T7 RNA polymerase to install poly(G) tails on mRNAs transcribed from polymerase chain reaction-generated DNA templates, we observed sharply increased synthesis of active proteins in vitro in coupled transcription/translation reactions. The ability of poly(G) tails to functionally stabilize transcripts from polymerase chain reaction-generated templates allows proteins encoded by translational open reading frames on genomic DNA or cDNA to be synthesized directly and efficiently in vitro.

Recovery of a Virulent Strain of Newcastle Disease Virus from Cloned cDNA: Expression of a Foreign Gene Results in Growth Retardation and Attenuation

A recombinant mesogenic NDV strain, Beaudette C, and an engineered recombinant NDV expressing an additional gene were generated entirely from cloned cDNAs. For this purpose, a full-length cDNA clone of the virus genome, represented in eight different subgenomic fragments, was assembled in a transcription plasmid between a T7 RNA polymerase promoter and a hepatitis delta virus ribozyme sequence. Infectious NDV could be generated in the cells infected with recombinant vaccinia virus, which expressed T7 RNA polymerase, by simultaneous expression of antigenome-sense NDV RNA from the full-length plasmid and NDV NP, P, and L proteins from cotransfected plasmids. Recombinant virus was then amplified and recovered, either after inoculation of transfection supernatant into the allantoic cavity of embryonated specific-pathogen-free eggs or after further passage in cell culture. Characterization of the recombinant NDV showed similarities in growth and pathogenicity to that of the parental wild-type virus. By using this system, a recombinant NDV containing a foreign gene encoding chloramphenicol acetyltransferase (CAT) was generated. To do this, the CAT transcription cassette containing the CAT open reading frame, flanked by NDV gene start and gene end sequence motifs, was inserted into the region between the HN and L genes of the full-length cDNA. This construct was then used in the generation of a recombinant NDV expressing CAT protein. The CAT gene was maintained stably for at least eight passages without any detectable loss of the gene from the recombinant. Generation of the recombinant virus, however, was associated with reduced plaque size, slower replication kinetics, and more than 100-fold decrease in yield. In addition, the virus showed an increase in mean death time for eggs and a lower intracerebral pathogenicity index in day-old chicks, implicating attenuation of the recombinant virus. Thus, introduction of an additional gene into the NDV genome represents a method to achieve growth retardation and attenuation. These results also indicate that NDV can be engineered to express foreign protein stably and can be manipulated in the future for use as a vaccine vector.

Association of a Promoter Polymorphism of Tumor Necrosis Factor-α with Subacute Cutaneous Lupus Erythematosus and Distinct Photoregulation of Transcription ,

Ultraviolet irradiation stimulates keratinocytes and dermal fibroblasts to release cytokines involved in apoptosis and immunomodulation, such as tumor necrosis factor-alpha and interleukin-1alpha. Recent work has associated the -308A polymorphism of the human tumor necrosis factor-alpha promoter with systemic lupus erythematosus and adverse outcomes in several infectious diseases. To explore the role of this polymorphism in ultraviolet-induced disease, we used two approaches. First, we examined its prevalence in individuals with different ultraviolet sensitivity. Compared with healthy controls, there was a substantially increased prevalence of -308A in subacute cutaneous lupus erythematosus, an extremely photosensitive form of cutaneous lupus erythematosus, but not in discoid lupus erythematosus, a less photosensitive form. Next, to examine molecular regulation by tumor necrosis factor -308A, cultured 3T3 fibroblasts were transiently transfected with chloramphenicol acetyl transferase reporter constructs under the control of either -308A or the wild-type -308G promoter. Without added interleukin-1alpha the two constructs produced similar baseline chloramphenicol acetyl transferase activity and similar responses to ultraviolet. The responses to interleukin-1alpha, a photoinduced cytokine, were markedly different: interleukin-1alpha without ultraviolet produced a 15-fold increase in chloramphenicol acetyl transferase transcription from the -308A construct without affecting the wild-type -308G. Interleukin-1alpha plus ultraviolet B caused a remarkable 300-fold increase in -308A chloramphenicol acetyl transferase transcription over baseline, while increasing the wild type to <15% of this level. These results indicate a clear difference between the two promoters, including a striking synergy between ultraviolet B and added interleukin-1alpha in the induction of transcription by the tumor necrosis factor-alpha -308A promoter. Overall, our findings indicate a strong linkage between the -308A polymorphism and subacute systemic lupus erythematosus, which is likely to directly contribute to the photosensitivity of these patients.

The 3'-untranslated region of the human estrogen receptor alpha gene mediates rapid messenger ribonucleic acid turnover.

Human estrogen receptor-alpha messenger RNA (hERalpha mRNA) has a relatively short half-life, which was determined to be approximately 5 h in MCF-7 cell line after actinomycin D treatment. The 3'-untranslated region (3'UTR) of hERalpha mRNA was previously shown to completely down-regulate chloramphenicol acetyltransferase activity when present at the 3'-end of chloramphenicol acetyltransferase transcripts, suggesting a destabilizing function of the hERalpha 3'UTR sequence. Chimeric genes composed of a serum-inducible Fos promoter, GH-coding sequences, and different segments of the hERalpha complementary DNA 3'UTR sequence were used to confirm this hypothesis and to localize the RNA region responsible for the destabilizing effect. The presence of the complete hERalpha 3'UTR reduced the half-life of the reporter mRNA from more than 24 to 3 h. When the hERalpha 3'UTR was subdivided into four fragments (UTR1-4), one fragment, UTR2, retained the most ability to down-regulate the reporter mRNA (t1/2 = 4 h). A stretch of four AUUUA motifs within UTR2 was shown not to mediate mRNA destabilization. In contrast, further subdivision of the UTR2 into three parts (UTR2a-c) resulted in the loss of the destabilizing activity. Finally, recombination of two UTR2 subfragments (UTR2a and -b) partially restored this function, indicating a cooperative role among the three UTR2a-c subfragments in the process that leads to destabilization of the hERalpha transcript.

Characterization of the 5'-flanking region of the human RNA-specific adenosine deaminase ADAR1 gene and identification of an interferon-inducible ADAR1 promoter.

The double-stranded RNA-specific adenosine deaminase (ADAR1) is inducible by interferon (IFN) and is implicated in the editing of viral RNAs during lytic and persistent infection. We have now isolated and characterized human genomic clones that contain the promoter region required for transcription of the ADAR1 gene. Rapid amplification of cDNA 5'-ends (5'-RACE) identified additional upstream exon 1 sequence that was localized on P1-phage and lambda-phage genomic clones by Southern gel-blot analysis and sequence analysis. A Northern gel-blot analysis using a probe corresponding to the 5'-RACE exon 1 sequence and adjacent exon 2 sequence detected a major RNA transcript of approximately 6.7kb that was IFN-inducible in human amnion U cells. Transient transfection assays, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the ADAR1 gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed a consensus copy of the IFN-Stimulated Response Element (ISRE) involved in IFN inducibility that was flanked by a Kinase Conserved Sequence (KCS)-like element previously found to be unique to the human and mouse PKR gene promoters. A 63-bp minimal promoter fragment possessing the KCS-like and ISRE elements was sufficient to drive IFN-inducible transcription.

Cloning and characterization of the promoter for the liver isoform of the rat carnitine palmitoyltransferase I (L-CPT I) gene.

Carnitine palmitoyltransferase I (CPTI) catalyses the transfer of long chain fatty acids to carnitine for translocation across the mitochondrial inner membrane. The cDNAs of two isoforms of CPT I, termed the hepatic and muscle isoforms, have been cloned. Expression of the hepatic CPT I gene (L-CPT I) is subject to developmental, hormonal and tissue specific regulation. We have cloned the promoter of the L-CPTI gene from a rat genomic library. In the L-CPTI gene, there are two exons 5' to the exon containing the ATG that initiates translation. Exon 1 and the 5' end of exon 2 contain sequences that were not previously described in the rat L-CPTI cDNA. There is an alternatively spliced form of the L-CPTI mRNA in which exon 2 is skipped. The proximal promoter of the L-CPTI gene is extremely GC rich and does not contain a TATA box. There are several putative Sp1 binding sites near the transcriptional start site. A 190 base pair fragment of the promoter can efficiently drive transcription of luciferase and CAT (chloramphenicol acetyltransferase) reporter genes transiently transfected into HepG2 cells. Sequences in both the first intron and the promoter contribute to basal expression. Our results provide the foundation for further studies into the regulation of L-CPTI gene expression.

Stimulation of troutCYP1A gene expression in mouse HEPA-1 cells by 3-methylcholanthrene

TroutCYP1A-CAT expression construct was generated by cloning approximately 3.5 Kb 5' flanking DNA of trout liverCYP1A gene in front of CAT gene at pCAT-basic vector. Hepa 1 cells, which are known to contain a functional arylhydrocarbon receptor' were transfected with troutCYP1A-CAT using lipofectin. 3-Methylcholanthrene (1 nM) was added into hepa 1 cells in culture in order to examine if 5' flanking DNA of troutCYP1A gene could interact with mouse transactivating factors to bring about transcription of the chloramphenicol acetyltransferase(CAT) reporter gene. The level of CAT protein was measured by CAT ELISA and the level of CAT mRNA was determined by RTPCR. The treatment of 1 nM 3-methylcholanthrene resulted in two fold increases in CAT protein as well as CAT mRNA compared to untreated control hepa 1 cells. These data indicate that arylhydrocarbon receptors of mouse hepa 1 cells are functional to activate exogenously transfected troutCYP1A-CAT construct in terms of both transcription and translation of CAT. We also examined the effect of 3-methylcholanthrene on endogeneouscyp1a1 activity in hepa 1 cell. 3-Methylcholanthrene (1 nM) treatment to hepa 1 cells transfected with troutCYP1A-CAT construct stimulated the level of cyp1a1 mRNA by two folds and the activity of ethoxyresorufin-O-deethylase by two fold compared to that of control cells. In this study we reported that troutCYP1A-CAT reporter gene expression construct could be expressed by 3-methylcholanthrene treatment in mouse hepa 1 cells. Thus troutCYP1A-CAT could serve as a good model to study the mechanism of regulation ofCYP1A1 gene expression.

Regulation of tissue inhibitor of metalloproteinase-1 in fibroblasts and acute phase proteins in hepatocytes in vitro by mouse oncostatin M, cardiotrophin-1, and IL-6.

Mouse oncostatin M (mOSM) has been recently cloned; however, its full spectrum of biologic functions has not been defined. To assess its potential role in inflammation, we have tested the activity of mOSM in vitro in regulation of fibroblasts and hepatic cells. At concentrations of 10 and 20 ng/ml, mOSM stimulates tissue inhibitor of metalloproteinase-1 (TIMP-1) mRNA in NIH-3T3 mouse embryonic fibroblasts, rat lung fibroblasts, and rat synovial fibroblasts, whereas mouse cardiotrophin-1 (mCT-1) or human OSM (hOSM) did not. Similarly, only mOSM was able to induce transcription of chloramphenicol acetyl-transferase (CAT) in NIH-3T3 cells transfected with a minimal TIMP-1 promoter/CAT construct. Mouse OSM had strong action inducing primary rat hepatocyte cultures to produce acute phase proteins; however, mOSM was very weak in its ability to stimulate acute phase protein synthesis in rat H35 cells or human HepG2 cells, which was consistent with weak STAT activation in H35 cells and HepG2 cells. Binding studies showed that NIH-3T3 cells possessed high affinity binding sites for mOSM, but rat H35 cells did not. On the other hand, mCT-1 and mouse IL-6 induced strong STAT activation as well as marked increases in acute phase protein production by H35 cells. These results indicate that mOSM does not share a functional receptor with mCT-1 or hOSM in mouse and rat cells and that hOSM does not activate the putatively specific OSM receptor on mouse or rat cells. These results also suggest that mOSM is an important cytokine in inflammation, through modulation of fibroblast function as well as hepatocyte responses.

Isolation of the Interferon-Inducible RNA-Dependent Protein KinasePkrPromoter and Identification of a Novel DNA Element within the 5′-Flanking Region of Human and MousePkrGenes

The RNA-dependent protein kinase (PKR) is inducible by interferon (IFN) and is implicated in the antiviral and antiproliferative actions of IFN. We have now isolated human genomic clones that contain the promoter region required for transcription of thePkrgene. Transient transfection analyses, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5′-flanking fragments of thePkrgene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed an element (5′GGAAAACGAAACT3′) involved in IFN inducibility that corresponds to the consensus sequence of the IFN-stimulated response element (ISRE). Comparison of the promoter sequence of the humanPkrgene to that of the mouse homolog identified a novel element (5′GGGAAGGCGGAGTCC3′) immediately upstream of the ISRE element which so far is unique to the human and mousePkrgene promoters. We have designated this new motif as KCS, for kinase conserved sequence. Deletion and substitution mutants of thePkrpromoter region showed that the ISRE element was required for transcriptional induction by type I IFN, whereas the KCS motif increased promoter activity mediated by the ISRE. Additional potential regulatory cis-elements were identified in the humanPkrpromoter that are commonly associated with growth control regulation and differentiation. Other than the ISRE and novel KCS elements, the overall organization of potential binding sites for transcription factors was not well conserved between the IFN-inducible promoters of the human and mousePkrgenes. The strict conservation of sequence, distance, and position of KCS, relative to ISRE, together with mutagenesis results, suggest an important functional role for the newly recognized KCS motif.

Translational regulation of angiotensin II type 1A receptor. Role of upstream AUG triplets.

The cDNA sequence of rat angiotensin II type 1A receptor (AT1AR) shows that AT1AR transcripts have AUG triplets in the 5'-leader region that may begin a short open reading frame encoding an 11-amino acid peptide. In this study, the mutational inactivation of the start codon of the short open reading frame in AT1AR-chloramphenicol acetyltransferase (CAT) reporter gene constructs resulted in a 2.6-fold increase in CAT activity, whereas CAT transcript levels were not affected. Furthermore, experiments with rat AT1AR cDNA-transfected Cos-7 cells revealed that mutagenesis of the upstream AUG increased the AT1AR protein up to 2.5-fold, although AT1AR transcript levels showed no changes. The synthetic peptide corresponding to the sequence of the short open reading frame significantly suppressed the amount of AT1AR product in the in vitro translation system. The inhibiting effect of the short open reading frame appears to operate at least in part at the level of translation initiation, because polysome analysis with transfected Cos-7 cells showed that mutagenesis of the upstream AUG resulted in a shift of AT1AR mRNA distribution from a smaller to larger fraction of polysomes. Taken together, these results show that the upstream AUG inhibits translational regulation, suggesting that the short open reading frame in the 5'-leader region of AT1AR transcripts has a certain role in the translation of AT1AR protein.

Dual function of the intron of the rat insulin I gene in regulation of gene expression.

Since the short intron in the 5'-untranslated region (5'-UTR) has been preserved during duplication of the insulin genes in rodents we postulated a possible involvement of these sequences in the regulation of gene expression. To examine this hypothesis we fused nested 5'-deletion fragments of the rat insulin I (rins1) promoter and sequences of the 5'-UTR up to nucleotide +170 with the reporter gene chloramphenicol acetyltransferase (CAT) and generated two series of expression constructs differing by the presence or absence of the intron (rins11VS). Transient expression of these chimeric genes in HIT M2.2.2 cells revealed a four-fold higher CAT expression in the presence of rins1IVS. Comparison of the CAT transcript quantities generated by both counterparts showed only a 1.7-fold difference in the total nuclear RNA fraction, but a four-fold difference in the fraction of nuclear polyadenylated RNA. Further analysis of cytoplasmic RNA excluded nuclear-cytoplasmic transport, RNA stability, and efficiency of translation as targets of the rins1IVS-mediated effect. The higher rate in polyadenylated CAT transcripts generated by rins1IVS-containing vectors suggests a possible coupling between splicing and polyadenylation. Transient expression studies using chimeras containing mutations or deletions between nucleotides -87 and +110 showed a reduction of expression by 30%. These data suggest a dual function of the rins1 intron on transcription initiation and transcript maturation.

Molecular and Microscopic Analysis of Altered Gene Expression in Transgenic and Gene Targeted Mice

Two myosin heavy chains (MyHC) are expressed in the mammalian heart and are differentially regulated during development. In the mouse, the α-MyHC is expressed constitutively in the atrium. At birth, the β-MyHC is downregulated and replaced by the α-MyHC, which is the sole cardiac MyHC isoform in the adult heart. We have employed transgenic and gene-targeting methodologies to study the regulation of cardiac MyHC gene expression and the functional and developmental consequences of alteredα-MyHCexpression in the mouse.We previously characterized an α-MyHC promoter capable of driving tissue-specific and developmentally correct expression of a CAT (chloramphenicol acetyltransferase) marker in the mouse. Tissue surveys detected a small amount of CAT activity in the lung (Fig. 1a). The results ofin situhybridization analyses indicated that the pattern of CAT transcript in the adult heart (Fig. 1b, top panel) is the same as that ofα-MyHC(Fig. 1b, lower panel). The α-MyHC gene is expressed in a layer of cardiac muscle (pulmonary myocardium) associated with the pulmonary veins (Fig. 1c). These studies extend our understanding ofα-MyHCexpression and delimit a third cardiac compartment.

Characterization of the Duffy Gene Promotor: Evidence for Tissue-Specific Abolishment of Expression in Fy(a−b−) of Black Individuals

We have previously identified a novel first exon of Duffy gene and two inverse GATA motifs in the 600 bp 5′ flanking region. The proximal GATA is positioned downstream from the start position of endothelium and upstream from that of erythroid. One base substitution (−365T→C) was found in the proximal GATA motif from three black Fy(a−b−) individuals, and was regarded as a common polymorphic mutation in black Fy(a−b−) individuals. The upstream sequence of the novel first exon was inserted in the upstream of chloramphenicol acetyltransferase (CAT) gene and transfected in human erythroleukemia cell line (HEL) and human microvascular endothelial cells (HMvEC). The black type mutation abolished the CAT transcription in HEL cells but not in HMvEC. Deletion mutagenesis study revealed that the proximal GATA motif represent the erythroid regulatory core region for Duffy gene. Gel shift assay showed that the proximal GATA motif is the target sequence of GATA-1. These studies indicate that the black type mutation abolishes Duffy gene expression in erythroid but not in postcapillary venule endothelium, which is compatible with the Northern blot and immunohistochemical observation in black Fy(a−b−) individuals.

Tumor suppressor p53 is a direct transcriptional activator of the human bax gene

The bax gene promoter region contains four motifs with homology to consensus p53-binding sites. In co-transfection assays using p53-deficient tumor cell lines, wild-type but not mutant p53 expression plasmids transactivated a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase. In addition, wildtype p53 transactivated reporter gene constructs containing a heterologous minimal promoter and a 39-bp region from the bax gene promoter in which the p53-binding site consensus sequences reside. Introduction of mutations into the consensus p53-binding site sequences abolished p53 responsiveness of reporter gene plasmids. Wild-type but not mutant p53 protein bound to oligonucleotides corresponding to this region of the bax promoter, based on gel retardation assays. Taken together, the results suggest that bax is a p53 primary-response gene, presumably involved in a p53-regulated pathway for induction of apoptosis.

Thyroid hormone receptors bind to the promoter of the mouse histone H10 gene and modulate its transcription.

It has been shown that the mouse histone H10 promoter contains a DNA element, composed of a direct repeat of the sequence GGTGACC separated by 7 nt, which is able to bind retinoic acid receptors and to modulate transcription of reporter genes following treatment with retinoic acid. We have now investigated whether this DNA motif is also responsive to thyroid hormone. We co-transfected CV-1 monkey kidney cells with chloramphenicol acetyltransferase (CAT) expression plasmids containing either 740 bp of the H10 wild-type promoter or five copies of the repeat element cloned in front of the thymidine kinase promoter and expression vectors for human thyroid hormone receptors (TRs) alpha or beta and retinoid X receptor alpha (RXR alpha). Treatment of transfected cells with triiodothyronine led to a dose-dependent increase in CAT activity. Transfection experiments with increasing amounts of expression vectors for either TR alpha or RXR alpha resulted in up to 6-fold enhancement of CAT transcription. Furthermore, point mutations within the half-sites of the response element of the H10 promoter, as well as deletions within the interspace region, lowered CAT activity to 60-80% of that of the wild-type control. Electrophoretic mobility shift assays showed that the repeat element was able to form retarded complexes with TR alpha homodimers, as well as with TR alpha-RXR alpha heterodimers. Our results suggest that thyroid hormone receptors are involved in the regulation of mouse histone H10 expression.

Down-regulation of Rous sarcoma virus long terminal repeat promoter activity by a HeLa cell basic protein.

We have previously isolated a HeLa cell cDNA encoding a 21-kDa polypeptide that is 48% similar to transcription factor IIS. To explore the possibility that p21 plays a role in transcriptional regulation in vivo, we tested the effect of p21 expression on the synthesis of reporter chloramphenicol acetyltransferase (CAT) in transfected COS-1 cells. CAT formation under control of the Rous sarcoma virus long terminal repeat (RSV LTR) promoter was decreased nearly 20-fold in cells coexpressing p21. In contrast, CAT production under control of other sequence elements was only slightly reduced (human immunodeficiency virus type 1 LTR, simian virus 40 early promoter), unaffected (human heat shock protein of 70-kDa promoter, adenovirus major late promoter TATA box), or increased (terminal deoxynucleotidyltransferase initiator element, c-fos promoter) by p21 coexpression as compared to cells cotransfected with the parental vector. The abundance of steady-state CAT transcripts from RSV LTR was also decreased by p21 expression in a dose-dependent manner, suggesting that transcription of RSV LTR/CAT is under negative control by p21. Consistent with an effect on transcription, p21 was localized in nuclei of transfected cells. Deletion analysis of p21 indicated that the sequences essential for inhibition of RSV LTR function include the previously identified ARg/Ser-rich region and zinc finger-like motif. Proliferation of chicken embryo fibroblasts transfected with an infectious molecular clone of RSV was diminished by p21 expression, which also resulted in fewer transformed foci.