Chloramphenicol Acetyltransferase Constructs Research Articles

The Gnb5 gene is a novel ?-transducin homolog transcribed from a divergent promoter located immediately upstream of the Syrian hamster p53 P1 promoter

Regulatory regions controlling p53 gene transcription in Syrian hamster embryo cells were characterized by use of chloramphenicol acetyl-transferase (CAT) constructs encompassing various subfragments of its 5'-flanking sequences. This analysis identified a 961 bp PstI-SacI (PS) fragment upstream from the p53 P1 promoter, which exhibited promoter activity only in the reverse orientation relative to the p53 gene. Northern hybridizations of mRNA from hamster embryo cells with genomic probes containing the PS fragment detected a 2.1-kb transcript expressed at much lower levels than the p53 mRNA. Steady-state levels of the 2.1-kb mRNA were threefold higher in actively growing cells than in cells from confluent cultures. Library screenings with PS-containing probes resulted in the isolation from exponentially growing cells of a cDNA, the nucleotide sequence of which showed no significant homology to genes previously described. This novel gene, named Gnb5, for guanine nucleotide-binding protein, beta 5, codes for a protein of 538 amino acids with a highly acidic amino terminus containing a proline-rich domain, followed by a neutral domain with five repeat units of the beta-transducin (WD-40) motif. The homology with beta subunits of G proteins and with other WD-40 repeat-containing proteins was restricted to the repeats. The Gnb5 gene is well conserved in rodents and primates, as the hamster Gnb5 cDNA recognized, under high stringency conditions, the human and mouse counterparts in Southern and Northern hybridizations. Expression of Gnb5 in adult tissues was detected preferentially in testes, in both hamsters and humans.

Identification of Two Regulatory Elements within the Promoter Region of the Mouse Connexin 43 Gene

To define the minimal sequences required for expression of the connexin 43 gene (cx43) in myometrial cells, we generated 5' deletion constructs of a fragment extending 1686 base pairs upstream and 162 base pairs downstream of the transcription start site and determined their ability to drive expression of the chloramphenicol acetyltransferase reporter gene in transfected myometrial cell lines. Our investigation revealed two cis-acting regulatory elements within this fragment. Deletion of a region extending from -102 to -92 led to an increase of the promoter activity by greater than 10-fold, indicating a presence of a repressor element. Deletion of a region extending from -72 to -62 caused a decrease of the promoter activity of a similar extent, implying the existence of a positive element. Electrophoretic mobility shift assays demonstrated that synthetic oligonucleotides derived from these two small regions can each bind with a nuclear protein(s) prepared from myometrial cells, and an introduction of three and two base substitutions into each of these oligomers was sufficient to abolish their protein binding capability. These same mutations, when incorporated in the chloramphenicol acetyltransferase constructs, diminished regulatory functions of the negative and positive elements, and the protein(s) that bind to these functional elements was found in several tissues known to express cx43 gene.

Nickel and skin irritants up-regulate tumor necrosis factor-alpha mRNA in keratinocytes by different but potentially synergistic mechanisms.

A critical role of tumor necrosis factor (TNF)-alpha in irritant contact dermatitis and in the challenge phase of allergic contact dermatitis has recently been demonstrated in vivo. As in situ hybridization studies have indicated that keratinocytes were the cellular source of TNF-alpha in these reactions, we studied the mechanisms of TNF-alpha mRNA induction in keratinocytes by agents that induce contact dermatitis. Murine la-/CD3- epidermal cells were stimulated with phorbol myristate acetate (PMA), dimethylsulfoxide (DMSO), sodium dodecyl sulfate (SDS) and NiSO4, all of which up-regulated epidermal cell TNF-alpha mRNA production. In contrast, trinitrobenzenesulfonic acid and trinitrochlorobenzene did not significantly up-regulate TNF-alpha mRNA. These results were confirmed with murine keratinocyte cell lines. In keratinocytes transfected with a chloramphenicol acetyltransferase construct containing the -1059 to +138 base pair TNF-alpha promoter, increased promoter activity was observed upon stimulation with PMA and DMSO. In addition, PMA stimulation did not affect the stability of TNF-alpha mRNA. The PMA- but also the DMSO- and SDS- induced up-regulation of TNF-alpha mRNA was abolished by an inhibitor of protein kinase C (PKC). In contrast, NiSO4 up-regulated TNF-alpha mRNA by a PKC-independent mechanism, did not increase TNF-alpha promoter activity, but markedly increased the stability of the TNF-alpha mRNA. Co-stimulation with PMA and NiSO4 induced a marked increase in TNF-alpha mRNA over that obtained with each agent alone. Thus, whereas PKC-dependent irritants act by up-regulating TNF-alpha promoter activity, nickel acts via post-transcriptional regulation. Our results also establish that some irritants and irritant sensitizers directly induce TNF-alpha in keratinocytes without intermediate Langerhans cell-derived signals.

A combination of distal and proximal regions is required for efficient prolactin regulation of transfected rabbit alpha s1-casein chloramphenicol acetyltransferase constructs.

In the rabbit, alpha s1-casein is the major casein secreted in the milk. Transcription of the alpha s1-casein gene is induced by PRL. To define the positions of the cis-sequences involved in the control of rabbit alpha s1-casein gene expression by PRL, chimeric genes containing upstream regions of alpha s1-casein gene linked to the chloramphenicol acetyltransferase gene were cotransfected into Chinese hamster ovary cells with the plasmid expressing the rabbit mammary PRL receptor. It was observed that a distal fragment -3442/-3118 was responsible for a high induction of PRL sensitivity when linked in the 5'-position to a chimeric construct (-391/1774)-chloramphenicol acetyltransferase. A cooperation between distal and proximal regions of the alpha s1-casein gene is responsible for the PRL-dependent enhancer activity of the distal fragment. The mammary gland-specific nuclear factor-like binding sequence found around position -90 in the proximal promoter of the alpha s1-casein gene is involved in this cooperation. The distal fragment was further studied to determine the position of regulatory regions. A -3442/-3385 fragment was sufficient to induce a PRL sensitivity similar to that conferred by the larger -3442/-3118 distal fragment, but multiple interactions are likely to exist between other regulatory regions included in this distal fragment. Four DNA-binding regions (I-IV) have been identified within the reduced -3442/-3385 fragment by footprint experiments using rabbit mammary gland or liver nuclear extracts (NE). Protected area III is observed using both NE. Protected areas I, II, and IV are specific for lactating mammary gland NE. The sequences of areas I and IV share several homologies with the sequence of the mammary gland-specific nuclear factor-binding site.

A combination of distal and proximal regions is required for efficient prolactin regulation of transfected rabbit alpha s1-casein chloramphenicol acetyltransferase constructs [published erratum appears in Mol Endocrinol 1995 Apr;9(4):412

A combination of distal and proximal regions is required for efficient prolactin regulation of transfected rabbit alpha s1-casein chloramphenicol acetyltransferase constructs [published erratum appears in Mol Endocrinol 1995 Apr;9(4):412

Characterization of retinoic acid- and cell-dependent sequences which regulate zif268 gene expression in osteoblastic cells.

We have previously shown that retinoic acid (RA) induces differentiation in an osteoblastic cell line derived from embryonic rat calvaria and that RA has selective effects on zif268 gene expression in these preosteoblastic cells,distinct from those in more mature osteoblasts. In this study we demonstrate that the RA-dependent transcriptional increase in zif268 gene expression is mediated by the interaction of RA receptors (RARs) with a 17 base pair sequence in the zif268 promoter containing a single half-site motif (GTTCA), identical to each of the direct repeats seen in the RAR beta 2 gene. The sequence appears relatively RA-specific, since the zif268 RA-responsive element is not activated by 1,25-dihydroxyvitamin D3 or thyroid hormone (T3). However, cotransfection of RAR expression vectors and an SV-40 promoter chloramphenicol acetyltransferase (CAT) construct containing the single zif268 RA-responsive motif into CV-1 cells demonstrates that the alpha-, beta-, and gamma-RARs transactivate through this element. Extensive mutagenesis of the zif268 promoter region containing the RA response element (RARE) motif confirms that the transactivation and nuclear protein binding activity of this region requires only the half-site motif. The direct involvement of RAR in this DNA-protein interaction has been demonstrated by competitive gel retardation analysis using consensus RAREs and super-shifting of the DNA-protein complex with mouse alpha- or gamma-RAR monoclonal antibodies. In addition, we found that cell-specific suppression of RA-stimulated zif268 gene expression can be attributed to a 29 base pair nucleotide sequence, located downstream of the RA-responsive region in the zif268 gene. This sequence appears to be bound specifically by nuclear protein(s) from several cell types, including osteoblasts. The presence of this sequence in cis to the zif268 RARE or the consensus beta RARE completely blocks the RA-responsiveness of the zif268 gene in differentiated osteoblasts. These data extend the broad spectrum of RA-responsive sequences necessary for DNA binding and transactivation to include regulation via single RARE half-site motifs and suggest that the lack of RA responsiveness in differentiated osteoblasts may be mediated by cell-specific suppression of gene expression.

Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.

In pancreatic beta-cells, the high Km glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake. Expression of the transporter has been reported to be modulated by glucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translation initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.

Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E.

Abstract Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B.

Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E.

Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B.

Activation of nuclear factor kappa B and oncogene expression by 12(R)-hydroxyeicosatrienoic acid, an angiogenic factor in microvessel endothelial cells.

12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an arachidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is due to a direct mitogenic effect on microvessel endothelial cells via yet to be elucidated cellular and molecular mechanisms. In the present study, we demonstrated the ability of 12(R)-HETrE to stimulate the growth of quiescent endothelial cells in a time- and concentration-dependent manner with a maximal effect at 0.1 nM. This effect was highly stereospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transfection experiments with chloramphenicol acetyltransferase constructs of oncogene promoter regions demonstrated significant increases over control (0.5% fetal calf serum) in c-myc-, c-jun, and c-fos mRNA levels and expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoretic mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides corresponding to known transcriptional binding sites, including AP-1, AP-2, SP1, TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kappa B and to a lesser extent, AP-1. No significant increase was observed in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, and c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in part, via the activation of NF kappa B and expression of these protooncogenes.

Thyroid-specific expression and cyclic adenosine 3',5'-monophosphate autoregulation of the thyrotropin receptor gene involves thyroid transcription factor-1.

The chimeric chloramphenicol acetyltransferase (CAT) construct, pTRCAT5'-199, containing the TSH receptor (TSHR) minimal promoter, -199 to -39 base pairs (bp), exhibits the thyroid specificity and TSH/cAMP autoregulation evident in TSHR gene expression. The present report shows that a cis-acting element between -189 and -175 bp, which binds thyroid transcription factor-1 (TTF-1), is involved in both activities. The 22 bp between -199 and -178 contains a positive element important for expression of the TSHR minimal promoter in rat FRTL-5 thyroid cells. DNAase I footprinting shows that extracts from functioning FRTL-5, but not non-functioning FRT thyroid or Buffalo rat liver (BRL) cells, protect a region between -189 and -175 bp. The protection is duplicated by TTF-1, and the protected element has only a two-base mismatch from the consensus TTF-1 element identified in the thyroglobulin (TG) and thyroid peroxidase minimal promoters. Gel mobility shift analyses reveal that FRTL-5 thyroid cell nuclear extracts form a specific protein/DNA complex with this region, which is prevented by the TTF-1 binding element from the TG promoter; FRT and BRL cell nuclear extracts do not have TTF-1 and do not form this complex. A role for the TSHR/TTF-1 binding element in thyroid-specific expression of the TSHR gene is evidenced as follows. Overexpression of TTF-1 in FRT or BRL cells, which have no TTF-1, increased the activity of pTRCAT5'-199, but not pTRCAT5'-177, which has no TTF-1 binding element. A nonsense mutation of the TTF-1 binding element eliminated TTF-1-induced activation of TSHR promoter activity in FRT or BRL cells and reduced TSHR promoter activity in FRTL-5 thyroid cells. In contrast, mutation of this element to the TTF-1 consensus sequence of the TG or thyroid peroxidase promoter had no significant influence on TSHR promoter activity. The activity of the TSHR/TTF-1 binding element requires a functioning cAMP response element (CRE). Thus, TTF-1 activity is lost when the CRE site is mutated to a nonfunctional, nonpalindromic sequence; it is, in contrast, maximized when CRE activity is maximized by its mutation to a consensus AP1 element. TTF-1 phosphorylation is important for binding and activity. Thus, binding of TTF-1 to the TSHR/TTF-1 element is phosphatase-sensitive and is increased by treating nuclear extracts with the catalytic subunit of protein kinase A. Overexpression of the catalytic subunit of PKA enhances TTF-1-increased activity of the TSHR minimal promoter.(ABSTRACT TRUNCATED AT 400 WORDS)

Interferon-beta induces metalloproteinase mRNA expression in human fibroblasts. Role of activator protein-1.

Matrix metalloproteinases are secreted enzymes important in inflammation and tumor invasion. Earlier, we demonstrated that in normal human FS-4 fibroblasts, collagenase and stromelysin mRNA levels are increased not only after treatment with known matrix metalloproteinase inducers such as tumor necrosis factor (TNF), interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate, but also with interferon-beta (IFN-beta). In this study, we compared the regulation of these matrix metalloproteinase genes by TNF and IFN-beta. We show that both TNF and IFN-beta increase steady-state levels of collagenase and stromelysin mRNAs with similar slow kinetics. The glucocorticoid dexamethasone blocked matrix metalloproteinase induction by both cytokines. The protein synthesis inhibitor cycloheximide inhibited collagenase mRNA induction by TNF or IFN-beta, suggesting that induction by both agents is indirect. Consistent with these observations, both TNF and IFN-beta increased c-fos and c-jun mRNA levels. Furthermore, treatment with TNF or IFN-beta increased the transcriptional activity of activator protein-1-responsive chloramphenicol acetyltransferase reporter gene constructs, including a native collagenase promoter-driven chloramphenicol acetyltransferase construct. These findings show that regulation of matrix metalloproteinase gene expression by both TNF and IFN-beta involves the transcription factor activator protein-1 and demonstrate a novel indirect mechanism of type I IFN-induced gene expression.

Basal and Tat-transactivated expression from the human immunodeficiency virus type 1 long terminal repeat in human placental trophoblast rules out promoter-enhancer activation as the partial block to viral replication

We have analysed the capacity of the trophoblast-derived malignant cell lines BeWo, JAR and JEG-3, and primary cultures of highly purified trophoblast cells to support the basal and Tat-mediated trans-activation-enhanced transcriptional activity of two distinct human immunodeficiency virus type 1 (HIV-1) isolates. Kinetic studies based on expression of long terminal repeat (LTR)-chloramphenicol acetyltransferase (CAT) constructs revealed that LTRs of both the prototype strain 3B and the highly cytopathic Zairean variant NDK were activated significantly in all target cells. Overall, the strongest activation was observed in primary trophoblasts. A novel modification of quantitative PCR was used to normalize LTR expression for transfection efficiency, enabling the calculation of specific expression rates in terms of muU CAT enzyme per fmol of transfected DNA. Using the latter criterion we determined that LTRs of both viruses were activated in decreasing order from trophoblasts to JAR, JEG-3 and BeWo cells; furthermore, the expression of HIV-1 3B LTR always significantly surpassed that of HIV-1 NDK. The effects of trans-activation on either of the LTRs, when assayed in cotransfection assays with various amounts of HIV-1 NDK-Tat expression vector, increased in a dose-dependent fashion and were comparable in a particular neoplastic cell line. Furthermore, the cell-specific LTR activity patterns did not correspond to the abundance of transcription factors binding specifically to the viral NF kappa B and SP1 motifs. Unlike SP1-binding proteins which were relatively abundant, substantially smaller amounts of proteins with NF kappa B specificity were found in all cells. Despite this apparent deficit in NF kappa B activity, trophoblasts supported a high basal activity of both LTRs. These data indicate that an insufficiency of basal or Tat-trans-activated LTR activity cannot account for the low level of HIV-1 replication in this important cell type.

Evaluation of the role of Ap1-like proteins in the enhanced apolipoprotein E gene transcription accompanying phorbol ester induced macrophage differentiation

Differentiation of THP1 monocytes to a macrophage phenotype is accompanied by increased apolipoprotein E gene transcription. Using transfection analysis with 5′ deletion mutations of the 5′ control region of the apo E gene in THP1 cells, we show that the −651 to +86 chloramphenicol acetyltransferase (CAT) construct is efficiently expressed in the monocyte; as has been reported for other cell types. Further, we found that an 176 bp region between −623 to −447 was required for the induction of apolipoprotein E gene transcription during 12- O-tetradecanoylphorbol-13-acetate-induced differentiation of monocytes to macrophages. Gel-retardation patterns of the apolipoprotein E promoter region using nuclear extracts from differentiated or undifferentiated THP1 cells revealed altered binding of Ap1-like nuclear factor/s to the −620 to −583 bp region after macrophage differentiation. Mutation of an Ap1 element at position −602 abolished specific binding of Ap1-like proteins to the −620 to −583 bp fragment of the apo E gene and significantly reduced expression of a −623 to +86 apo E-CAT construct during differentiation. These data indicate that differentiation-related expression of the apolipoprotein E gene following phorbol ester stimulation is transduced by gene elements between −623 and −447. Furthermore, the data indicate that transcriptional activation of the apo E gene during macrophage differentation is associated with induction of Ap1-like proteins which bind to the Ap1 response element present at −602 in the apolipoprotein E gene and importantly contribute to enhanced gene expression.

Phagocytosis of Mycobacterium tuberculosis modulates human immunodeficiency virus replication in human monocytic cells.

Macrophage activation resulting from phagocytosis has the potential to modulate human immunodeficiency virus (HIV) replication. We have determined the effects of phagocytosis of particulate stimuli on transcription and release of HIV. Using THP-1 and Mono Mac 6 human monocytic cell lines transfected with HIV long terminal repeat sequence chloramphenicol acetyltransferase (LTR CAT) constructs we have demonstrated that phagocytosis of Mycobacterium tuberculosis enhanced HIV-1 and -2 LTR CAT expression. However phagocytosis of zymosan or inert latex beads had little or no effect on CAT expression. Enhancement of HIV LTR CAT expression was dependent upon intact NF-kappa B binding sites and was independent of tumour necrosis factor alpha secretion. M. tuberculosis strains of different degrees of virulence induced similar levels of enhanced CAT expression. In contrast, phagocytosis of M. tuberculosis by HIV-1-infected THP-1 cells reduced supernatant reverse transcriptase (RT) activity without suppression of p24 antigen release. Phagocytosis of zymosan granules or latex particles did not alter released RT activity. However, phagocytosis of either M. tuberculosis, zymosan granules or latex particles by HIV-1-infected human peripheral blood monocyte-derived macrophages reduced supernatant RT activity. These data indicate that phagocytosis of M. tuberculosis may enhance HIV transcription in monocytic cells although it may reduce release of intact HIV.

The rat and human hemopexin genes contain an identical interleukin-6 response element that is not a target of CAAT enhancer-binding protein isoforms.

Hemopexin (Hx) is an abundant acute-phase protein (APP) that binds heme with high affinity. In rat hepatic cells, the transcription rate of the Hx gene is increased by interleukin (IL)-1 and IL-6. To investigate the cis-acting regulatory elements (REs) responsive to these hormones, chloramphenicol acetyltransferase constructs of rat and human Hx gene sequences were tested in transiently transfected hepatoma cells. An IL-6-RE was identified in the promoter of both rat and human Hx genes, the function of which was dependent on the core sequence (CCGGGAA) common in other APP genes. The previously characterized Hx A element mediated a relatively minor cytokine response as compared with the Hx IL-6-RE. The human Hx A element, in contrast to the rat and human Hx IL-6-REs, was strongly trans-activated by cotransfected CAAT enhancer-binding proteins (C/EBP)-beta and -delta. The rat gene homolog of the human Hx A element was inactive as a cytokine RE and was minimally trans-activated by C/EBP isoforms. Results of electrophoretic mobility shift assays indicated that the Hx IL-6-RE is a binding site for the IL-6-inducible nuclear protein IL-6 RE-BP, which also binds to the conserved IL-6-REs of other APP genes and is distinct from C/EBP beta.

17 beta-Estradiol inhibits expression of human interleukin-6 promoter-reporter constructs by a receptor-dependent mechanism.

We previously reported that 17 beta-estradiol inhibits cytokine-stimulated bioassayable IL-6 and the steady-state level of IL-6 mRNA. To determine the molecular basis of this effect, the transient expression of chloramphenicol acetyltransferase (CAT) reporter plasmid driven by the human IL-6 promoter was studied here in HeLa or murine bone marrow stromal cells (MBA 13.2). 17 beta-estradiol (10(-8) M) completely suppressed stimulated CAT expression in HeLa cells cotransfected with IL-6/CAT constructs and a human estrogen receptor (hER) expression plasmid; but had no effect on reporter expression in HeLa cells not transfected with hER. 17 beta-estradiol also inhibited stimulated expression in MBA 13.2 cells (which express the estrogen receptor constitutively) without the requirement of cotransfection of the hER plasmid. The hormonal effects were indistinguishable between constructs containing a 1.2-kb fragment of the 5' flanking region of the IL-6 gene or only the proximal 225-bp fragment. However, yeast-derived recombinant hER did not bind to the 225-bp segment in DNA band shift assays, nor did the 225-bp fragment compete for binding of an estrogen response element oligonucleotide to yeast-derived estrogen receptor. These data suggest that 17 beta-estradiol inhibits the stimulated expression of the human IL-6 gene through an estrogen receptor mediated indirect effect on the transcriptional activity of the proximal 225-bp sequence of the promoter.

Identification and characterization of a human herpesvirus 6 gene segment capable of transactivating the human immunodeficiency virus type 1 long terminal repeat in an Sp1 binding site-dependent manner

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) is transactivated by various extracellular signals and viral cofactors that include human herpesviruses. These transactivators are capable of transactivating the HIV-1 LTR through the transactivation response element, NF-kappa B, or other regulatory binding elements. Human herpesvirus 6 (HHV-6) is a potential cofactor of HIV-1. Here, we report that an HHV-6 gene segment, ZVH14, which can neoplastically transform NIH 3T3 and human keratinocytes, is capable of transactivating HIV-1 LTR chloramphenicol acetyltransferase constructs in an Sp1 binding site-dependent manner. Transactivation increased synergistically in the presence of multiple Sp1 sites and was dramatically reduced by cotransfection with oligomers designed to form triplex structures with HIV-1 LTR Sp1 binding sites. HIV-1 LTR NF-kappa B sites were not essential for ZVH14-mediated transactivation. A putative open reading frame in ZVH14, B115, which may encode a highly basic peptide consisting of 115 amino acid residues, showed transactivation capacity similar to that of ZVH14. This open reading frame also transactivated the HIV-1 LTR in an Sp1 site-dependent fashion in African green monkey kidney cells and human T cells. These data suggest that HHV-6 may stimulate HIV-1 replication via transactivation of Sp1 binding sites present in the HIV-1 promoter.

Isolation and Characterization of a TATA-less Promoter for the Human β3 Integrin Gene

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5' to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3' intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5' to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a "promoter-less" control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5' beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.

Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene

Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5′ to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3′ intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5′ to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a “promoter-less” control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5′ beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.