Chimeric Chloramphenicol Acetyltransferase Research Articles

SP-A 3'-UTR is involved in the glucocorticoid inhibition of human SP-A gene expression.

The synthetic glucocorticoid dexamethasone has a major inhibitory effect on human surfactant protein A1 (SP-A1) and SP-A2 gene expression that occurs at both the transcriptional and posttranscriptional levels. Toward the identification of cis-acting elements that may be involved in the dexamethasone regulation of SP-A mRNA stability, chimeric chloramphenicol acetyltransferase (CAT) constructs that contained various portions of SP-A1 or SP-A2 cDNA in place of the native CAT 3'-untranslated region (UTR) were transiently transfected into the lung adenocarcinoma cell line NCI-H441. CAT activity was reduced in NCI-H441 cells by exposure to 100 nM dexamethasone only for the chimeric CAT constructs that contained the SP-A 3'-UTR. Moreover, the inhibitory response seen with dexamethasone was greater for the 3'-UTR derived from the SP-A1 allele 6A3 than with the 3'-UTR derived from either the SP-A1 allele 6A2 or SP-A2 allele 1A0, indicating differential regulation between SP-A genes and/or alleles.

Cutting Edge: Clustered AU-Rich Elements Are the Target of IL-10-Mediated mRNA Destabilization in Mouse Macrophages

In the present study we show that IL-10-mediated inhibition of inflammatory gene expression can be mediated by an AU-rich element (ARE) cluster present in the 3' untranslated region (3'UTR) of sensitive genes. A series of chloramphenicol acetyl transferase (CAT) reporter gene constructs were prepared in which different fragments from the IL-10-sensitive KC mRNA 3'UTR were placed downstream of the coding region of the reporter gene CAT. CAT mRNA containing the KC 3'UTR was markedly destabilized as compared with the control CAT mRNA, and the decay rate was further increased in cells stimulated with IL-10. The KC 3'UTR contains an ARE cluster and three isolated ARE motifs. The ARE cluster spanning nucleotides 378-399 appeared to be both necessary and sufficient to mediate sensitivity to IL-10 because a 116-nucleotide fragment that contains the cluster conferred sensitivity, while mutation of the sequence between positions 378 and 399 eliminated sensitivity. The destabilizing effect of IL-10 was relatively selective, as the stability of chimeric CAT mRNAs was not modulated in cells treated with IFN-gamma or IL-4.

Mutational analyses of a type 2 peroxisomal targeting signal that is capable of directing oligomeric protein import into tobacco BY‐2 glyoxysomes

In this study of the type 2 peroxisomal targeting signal (PTS2) pathway, we examined the apparent discontinuity and conservation of residues within the PTS2 nonapeptide and demonstrated that this topogenic signal is capable of directing heteromultimeric protein import in plant cells. Based on cumulative data showing that at least 26 unique, putative PTS2 nonapeptides occur within 12 diverse peroxisomal-destined proteins, the current (-R/K-L/V/I-X5-H/Q-L/A-) as well as the original (-R-L-X5-H/Q-L-) PTS2 motif appear to be oversimplified. To assess the functionality of residues within the motif, rat liver thiolase (rthio) and various chimeric chloramphenicol acetyltransferase (CAT) proteins were expressed transiently in suspension-cultured tobacco (Nicotiana tabaccum L.) cv Bright Yellow cells (BY-2), and their subcellular location was determined by immunofluoresence microscopy. Hemagglutinin (HA)-epitope-tagged-CAT subunits, lacking a PTS2 (CAT-HA), were 'piggybacked' into glyoxysomes by PTS2-bearing CAT subunits (rthio-CAT), whereas signal-depleted CAT-HA subunits that were modified to prevent oligomerization did not import into glyoxysomes. These results provided direct evidence that signal-depleted subunits imported into peroxisomes were targeted to the organelle as oligomers (heteromers) by a PTS2. Mutational analysis of residues within PTS2 nonapeptides revealed that a number of amino acid substitutions were capable of maintaining targeting function. Furthermore, functionality of residues within the PTS2 nonapeptide did not appear to require a context-specific environment conferred by adjacent residues. These results collectively suggest that the functional PTS2 is not solely defined as a sequence-specific motif, i.e. -R/K-X6-H/Q-A/L/F-, but defined also by its structural motif that is dependent upon the physiochemical properties of residues within the nonapeptide.

Post-transcriptional Regulation of Erythropoietin mRNA Stability by Erythropoietin mRNA-binding Protein

We have previously identified a sequence in the 3'-untranslated region (3'-UTR) of erythropoietin (Epo) mRNA which binds a protein(s), erythropoietin mRNA-binding protein (ERBP). A mutant lacking the ERBP binding site (EpoM) was generated. Hep3B cells were stably transfected with a wild-type Epo (EpoWT) cDNA or EpoM cDNA construct located downstream of a promoter of cytomegalovirus. Following inhibition of transcription, the half-lives of EpoWT and EpoM mRNAs were 7 h and 2.5 h in normoxia, respectively. The EpoM mRNA half-life remained unchanged in hypoxia. EpoWT mRNA half-life increased approximately 40% in response to a 6-h hypoxic pre-exposure and an additional approximately 50% when pre-exposed to 12 h hypoxia. The steady-state level of EpoWT mRNA was 4-fold that of EpoM mRNA reflecting the difference in mRNA decay rates in normoxia. The Epo protein level expressed from exogenous EpoM was unchanged in both normoxia and hypoxia. In contrast, the Epo protein level expressed from exogenous EpoWT increased 50% in hypoxia when compared with normoxia. These observations were further supported by chimeric chloramphenicol acetyltransferase and Epo-3'-UTR constructs. We have demonstrated that Epo mRNA stability was modulated in normoxia and further by hypoxia, therefore, providing evidence that Epo is regulated at the post-transcriptional level through ERBP complex formation.

Chimeric constructs show that the unique N-terminal domain of the cyclic AMP phosphodiesterase RD1 (RNPDE4A1A; rPDE-IVA1) can confer membrane association upon the normally cytosolic protein chloramphenicol acetyltransferase.

A novel plasmid was generated which allowed the expression of the cytosolic bacterial enzyme chloramphenicol acetyl transferase (CAT) in COS-7 cells. Upon transfection, the majority of the novel CAT activity was found in the cytosol fraction of COS cells. Chimeric molecules were made between N-terminal portions of the type IVA cyclic AMP-specific rat 'dunce-like' phosphodiesterase (RD1) (RNPDE4A1A; rPDE-IVA1) fused to CAT at its N-terminus. Expression in COS-7 cells of chimeras formed from 1-100RD1-CAT and 1-25RD1-CAT now showed CAT activity associated with the membrane fraction. In contrast, a chimera formed from 26-100RD1-CAT showed an identical expression pattern to native CAT, with the major fraction of CAT activity occurring in the cytosol fraction. Membrane-bound CAT activity provided by 1-100RD1-CAT and 1-25RD1-CAT was not released by either high-salt or washing treatments but was solubilized in a dose-dependent fashion by the non-ionic detergent Triton X-100. Subcellular fractionation of COS-7 cells showed that, as with RD1, the membrane-bound activity of the RD1-CAT chimera followed that of the plasma membrane marker 5'-nucleotidase. Plasmids containing chimeric cDNAs were exposed to a coupled transcription-translation system that, in addition to the full-length chimeras, was found to generate a range of N-terminal truncated species due to initiation at different methionine residues. Incubation of the mature protein products formed in this system with a COS cell membrane fraction showed that only those chimeric CAT constructs containing the first 25 amino acids of RD1 became membrane-associated. The unique 25 amino acid N-terminal domain of RD1 contains structural information that can confer membrane association upon an essentially soluble protein.

Identification of a cyclic adenosine 3',5'-monophosphate-response element in the rat aromatase promoter that is required for transcriptional activation in rat granulosa cells and R2C leydig cells.

Cytochrome P450 aromatase, which converts testosterone to estradiol, is transcriptionally induced by FSH and cAMP during ovarian follicular development. At least one promoter element [-82/-31 base pairs (bp)] required for stimulation of the rat gene in granulosa cells binds steroidogenic factor-1, an orphan steroid receptor. In this paper, we demonstrate that an additional region, -161/-138 bp is required for cAMP regulation. This region shares homology with promoter sequences in the bovine 21-hydroxylase and mouse 11 beta-hydroxylase genes that are also induced by cAMP, yet each binds different proteins in granulosa cell nuclear extracts. The aromatase -161/-138 bp region contains a cAMP-response element (CRE)-like sequence, TGCACGTCA. Deletion or mutation of this sequence reduces promoter activity of chimeric chloramphenicol acetyl transferase (CAT) reporter constructs that are transiently transfected into granulosa cells and R2C Leydig cells. Granulosa cell nuclear proteins and R2C cell nuclear proteins specifically bind the -161/-138 bp region and form three protein/DNA complexes. Recombinant CRE-binding protein (CREB) binds the CRE-like sequence and forms a single band, and a CREB antibody retards the migration of CREB and one granulosa cell protein-aromatase DNA binding complex. Using Western blot analysis, CREB was demonstrated in granulosa cell nuclear extracts from all stages of follicular development. Thus, aromatase is transcriptionally regulated by a hexameric sequence binding SF-1 and a CRE sequence binding CREB and other factors present in granulosa cells and in R2C Leydig cells. The presence of identical SF-1 and CRE-like sequences in the human ovarian aromatase promoter II suggests that the human promoter may also be regulated in a similar manner.

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)

Characterization of a mitogen-response unit in the mouse lactoferrin gene promoter.

Lactoferrin is present in a variety of tissues and biological fluids; however, the amount differs significantly due to differential expressions. We have previously demonstrated that the mouse lactoferrin gene is regulated by estrogen through an estrogen-response DNA element located at -349, upstream from the transcription start site (+1). In this report, we characterized by deletion and mutation analyses a cluster of mitogen-response elements located between -80 and -40 of the mouse lactoferrin promoter. We demonstrated that the chimeric chloramphenicol acetyltransferase reporter constructs (the -103 to +1 sequence of the mouse lactoferrin gene) containing the mitogen-response unit of the lactoferrin gene were stimulated by cAMP, forskolin, 12-O-tetradecanoylphorbol-13-acetate, and epidermal growth factor/recombinant transforming growth factor-alpha (EGF/TGF-alpha) in a time- and dose-dependent manner. The sequence at position -52 to -40 (mLF-CRE) of the gene conferred transcriptional activation in the presence of forskolin, cyclic AMP, and 12-O-tetradecanoylphorbol-13-acetate in transiently transfected human endometrium carcinoma RL95-2 cells, whereas the region at -80 to -60 responded to EGF/TGF-alpha stimulation. Overexpression of the catalytic unit of protein kinase C or protein kinase A in the RL95-2 cells elevated the chloramphenicol acetyl-transferase activity of the reporter construct 5-6-fold. The mobility shift assay suggested that AP1 and CREB or related proteins participated in complex formation with the mLF-CRE, whereas different proteins bound to the EGF/TGF-alpha-response element.

Structural and functional characterization of the human decorin gene promoter. A homopurine-homopyrimidine S1 nuclease-sensitive region is involved in transcriptional control.

Decorin is a leucine-rich, chondroitin/dermatan sulfate proteoglycan which binds collagen and growth factors. We have recently completed the genomic organization of human decorin and discovered two alternatively spliced leader exons, designated exon Ia and Ib, in the 5'-untranslated region. Initial analysis of the sequences upstream to these two exons showed that promoter Ia contained only two GC boxes while promoter Ib contained a CAAT and two TATA boxes in close proximity to the transcription start site. To determine if these 5'-flanking sequences exhibited promoter activity, chimeric chloramphenicol acetyltransferase expression plasmids containing the promoter region of either exon Ia or Ib were transfected into HeLa and MG-63 osteosarcoma cells. The results showed that only the region flanking exon Ib was functional. In vitro transcription assay generated two transcripts of 92 and 82 base pairs (bp) indicating that both TATA boxes could be used. Using stepwise 5' deletion analysis we found that the minimum promoter region at -140 bp from the transcription start site, which contained only the CAAT and the two TATA boxes, exhibited strong promoter activity. When a larger construct containing an additional 800 bp of upstream region was tested, a significant increase in transcriptional activity was observed. Interestingly, this promoter region contained several putative binding sites for ubiquitous factors (AP1, AP5, and NF-kappa B) and for transforming growth factor-beta and a 150-bp homopurine/homopyrimidine element with several mirror repeats. When contained in a supercoiled plasmid, this sequence exhibited sensitivity to endonuclease S1, an enzyme that preferentially digests single-stranded DNA. Precise S1 mapping, obtained by direct sequencing of nine distinct S1-generated clones, revealed that in all cases the borders of the sensitive sequence resided within the pur/pyr segment. We propose that this region of the promoter could adopt an intramolecular hairpin triplex structure in vivo and may play a role in the chromatin organization at the decorin gene locus. In addition, this region was able to up-regulate a minimal heterologous promoter in transient transfection assays. The results show that the structure of the decorin gene promoter is different from that of any other proteoglycan promoter characterized so far and indicate that the pur/pyr segment plays a role in the regulation of gene transcription.

5' sequences of rubella virus RNA stimulate translation of chimeric RNAs and specifically interact with two host-encoded proteins.

Sequences at the 5' and 3' ends of the rubella virus (RV) genomic RNA can potentially form stable stem-loop (SL) structures that are postulated to be involved in virus replication. We have analyzed the function of these putative SL structures in RNA translation by constructing chimeric chloramphenicol acetyltransferase (CAT) RNAs, flanked either by both 5'- and 3'-terminal sequence domains from the RV genome or several deletion derivatives of the same sequences. After in vitro transcription of chimeric RNAs, the translational efficiencies of these RNAs were compared by the rabbit reticulocyte lysate translation system. For in vivo translation studies, the level of CAT activity was measured for chimeric RV/CAT RNAs expressed in transfected cells by the adenovirus major late promoter. Both in vivo and in vitro translation activities of the chimeric RNAs revealed that the presence of 5' and 3' SL sequences of RV RNA, in correct (+) orientation and context [5'(+)SL and 3'(+)SL, respectively] was necessary for efficient translation of chimeric RV/CAT RNAs. The presence of the RV 5'(+)SL sequence had the primary enhancing effect on translation. To identify host proteins which interact with the 5'(+)SL which may be involved in RV RNA translation, RNA gel-shift and UV cross-linking assays were employed. Two host proteins 59 and 52 kDa in size, present in cytosolic extracts from both uninfected and RV-infected cells, specifically interacted with the RV 5'(+)SL RNA. Direct binding comparisons between wild-type and mutant 5'(+)SL RNAs demonstrated that sequences in and around the bulge region of the terminal stem domain of this structure constituted a protein binding determinant. Human serum, qualified for anti-Ro/SS-A antigen specificity, immunoprecipitated 59- and 52-kDa protein-RNA complexes containing the RV 5'(+)SL RNA. However, poly- and monoclonal antisera raised against the recombinant 60- and 52-kDa Ro proteins failed to precipitate complexes containing the 5'(+)SL RNA. The identity of the proteins binding this RV cis-acting element remains to be determined; however, their role in RV translation is discussed.

Two 3',5'-cyclic-adenosine monophosphate response elements in the promoter region of the human gastric inhibitory polypeptide gene.

Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the human gastric inhibitory polypeptide (GIP) promoter into hamster insulinoma (HIT T15) cells indicated that the region between -180 and +14 is sufficient for basal promoter activity. Two CRE-BP1 binding sites were identified in this promoter region by DNase I footprinting with the bacterially expressed cAMP response element (CRE) binding protein, CRE-BP1. Mutation analyses showed that these two CREs are required for the basal promoter activity, and furthermore that one site, at nucleotide-158, contributed mainly to the cAMP inducibility of the GIP promoter in HIT T15 cells. Interestingly, the GIP promoter activity was repressed by the c-jun proto-oncogene product, possibly through the CREs.

Transcriptional repression of interleukin-6 gene by adenoviral E1A proteins.

Transcription of interleukin-6 (IL-6) gene in human HepG2 and HeLa cells was induced by treatment with interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), phorbol 12-myristate 13-acetate, or dibutyryl cyclic AMP. These agents enhanced the expression of chloramphenicol acetyltransferase (CAT) activity in cells transfected with chimeric CAT genes driven by the transcriptional regulatory regions of human IL-6 gene. Both induced and basal levels of CAT expression were severely repressed upon co-transfection of expression vectors encoding the adenoviral E1A289R or E1A243R protein. The conserved region 1 of E1A proteins was required for this activity. IL-6-CAT expression could also be induced by co-transfecting expression vectors containing cDNAs of the catalytic subunit of protein kinase A or c-jun. E1A repressed transcriptional induction by these agents as well. Similar inhibition was observed when a CAT gene driven by the NF kappa B element of the IL-6 gene was used as a reporter plasmid. In a cell line stably transfected with the E1A gene, IL-1 or TNF-alpha failed to induce IL-6 mRNA. Electrophoretic mobility shift assays were carried out with nuclear extracts of these cells using, as probes, the NF kappa B element or the multiple regulatory element of the IL-6 gene. With either probe, additional faster migrating DNA-protein complexes were formed in the extracts of E1A-expressing cells as compared with the extracts of the corresponding control cells. Experiments with NF kappa B antibody revealed differences between the different DNA-protein complexes formed in the extract of E1A-expressing cells. These observations suggest that E1A represses IL-6 gene transcription by interfering with the formation of appropriate DNA-protein complexes.

Tumor necrosis factor alpha and interferon gamma synergistically induce interleukin 8 production in a human gastric cancer cell line through acting concurrently on AP-1 and NF-kB-like binding sites of the interleukin 8 gene.

Interleukin 8 (IL-8) is a novel cytokine which possesses neutrophil chemotactic and activating activities in addition to chemotactic activity for basophils and T lymphocytes. It has been shown that IL-8 is produced by a variety of human somatic cells including monocytes/macrophages, dermal fibroblasts, vascular endothelial cells, keratinocytes, mesangeal cells, and several types of tumor cell lines. We have examined here whether or not human gastric cancer cell lines produce IL-8 in vitro. The production of IL-8 protein was detected by enzyme-linked immunosorbent assay in the culture supernatants derived from eight of nine human gastric cancer cell lines stimulated with either interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), or TNF alpha plus interferon gamma (IFN gamma). In some of the gastric cancer cell lines such as MKN 45 and KATO, TNF alpha plus IFN gamma synergistically induced the production of IL-8. In MKN 45 cells, synergistic increase of the steady state level of IL-8 mRNA by TNF alpha plus IFN gamma was not inhibited by cycloheximide treatment. Scatchard analysis revealed that IFN gamma changed neither the number nor the affinity constant of TNF alpha binding sites on a gastric cancer cell line, suggesting that the synergism was a post-receptor event. Furthermore, synergistic induction of chloramphenicol acetyltransferase activity by TNF alpha plus IFN gamma was observed in MKN 45 that were transiently transfected with chimeric chloramphenicol acetyltransferase reporter genes driven by the transcriptional regulatory region of human IL-8 gene. Through the mutation of the regulatory region of the IL-8 gene, both AP-1- and NF-kB-like factor binding elements were presumed to be involved in conferring the responsiveness to TNF alpha plus IFN gamma. Moreover, gel retardation analyses revealed that TNF alpha and IFN gamma synergistically induced the binding of NF-kB like as well as AP-1 like proteins bound to these sites. These results indicated that IFN gamma synergistically enhanced TNF alpha-induced IL-8 production in a human gastric cancer cell line through synergistic activation of transcription factors without up-regulating TNF alpha receptor.

Genetic element from human surfactant protein SP-C gene confers bronchiolar-alveolar cell specificity in transgenic mice

Transgenic mice bearing chimeric genes consisting of 5'-sequences derived from the human surfactant protein C (SP-C) gene and the bacterial chloramphenicol acetyltransferase (CAT) gene were generated. Analysis of CAT activity was utilized to demonstrate tissue-specific and developmental expression of chimeric genes containing 3.7 kb of sequences from the human SP-C gene. Lung-specific expression of the 3.7 SP-C-CAT transgene was observed in eight distinct transgenic mouse lines. Expression of the 3.7 SP-C-CAT transgene was first detected in fetal lung on day 11 of gestation and increased dramatically with advancing gestational age, reaching adult levels of activity before birth. In situ hybridization demonstrated that expression of 3.7 SP-C-CAT mRNA was confined to the distal respiratory epithelium. Antisense CAT hybridization was detected in bronchiolar and type II epithelial cells in the adult lung of the 3.7 SP-C-CAT transgenic mice. In situ hybridization of four distinct 3.7 SP-C-CAT transgenic mouse lines demonstrated bronchiolar-alveolar expression of the chimeric CAT gene, although the relative intensity of expression at each site varied within the lines studied. Glucocorticoids increased murine SP-C mRNA in fetal lung organ culture. Likewise, expression of 3.7 SP-C-CAT transgene increased during fetal lung organ or explant culture and was further enhanced by glucocorticoid in vitro. The 5'-regions of human SP-C conferred developmental, lung epithelial, and glucocorticoid-enhanced expression of bacterial CAT in transgenic mice. The increased expression of SP-C accompanying prenatal lung development and exposure to glucocorticoid is mediated, at least in part, at the transcriptional level, being influenced by cis-active elements contained within the 5'-flanking region of the human SP-C gene.

Inhibition of interferon-inducible gene expression by adenovirus E1A proteins: block in transcriptional complex formation.

Infection with wild-type adenovirus 5, but not with a mutant lacking the E1A gene, prevented the induction by interferon (IFN) alpha of chloramphenicol acetyltransferase (CAT) activity in HeLaM cell lines that had been permanently transfected with chimeric CAT reporter genes driven by the transcriptional regulatory regions of the IFN-responsive genes 561 and 6-16. Similar inhibition of IFN-inducible CAT activity was observed in cells that were cotransfected with the same reporter genes and plasmids expressing either the E1A 289- or 243-amino acid protein. These proteins also prevented the induction of CAT activity by IFN-gamma from a cotransfected HLA-DR alpha-CAT gene. Experiments with E1A mutants mapped the inhibitory activity to amino acid residues 38-65 of these proteins. In a HeLa cell line permanently expressing the E1A 289-amino acid protein, the replication of vesicular stomatitis virus and encephalomyocarditis virus was not inhibited by IFN-alpha, suggesting a global blockade of IFN responses. In accord with this theory, induction of 561, 1-8, and (2'-5')oligoadenylate synthetase mRNAs by IFN was blocked in these cells at the transcriptional level. The observed transcriptional inhibition could be attributed to the lack of formation of the crucial IFN-stimulated gene factor 3 (ISGF3) transcriptional complex. As shown by mobility shift assays, this complex was not formed in the nuclear extracts of IFN-treated adenovirus-infected cells or IFN-treated E1A-producing cells. These nuclear extracts were deficient in both ISGF3 alpha and ISGF3 gamma subunits. However, they did not block the formation of ISGF3 complex from exogenously added components.

A cluster of four Sp1 binding sites required for efficient expression of the human insulin receptor gene

Fragments of 5'-flanking sequences of the human insulin receptor gene were analyzed in transient expression assays after transfection of cell lines with expression assays after transfection of cell lines with an improved low background chloramphenicol acetyl-transferase vector system pSVOOCAT (Araki, E., Shimada, F., Shichiri, M., Mori, M., and Ebina, Y. (1988) Nucleic Acids Res. 16, 1627). Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions and insertions of the promoter of HIR gene into CHO and COS cells indicated that the region between -629 and -1 (initiator ATG is +1) is sufficient for maximal promoter activity. The DNA element of the cluster of four G-C boxes (-593 to -618) enhanced the transcription, examined by the low background pSVOOCAT vector system in vivo. DNase I footprinting and gel retardation experiments using partially purified LacZ-Sp1 hybrid proteins showed that the transcription factor Sp1 can bind to the cluster of the four G-C boxes of the promoter. Thus, the efficient expression of the human insulin receptor gene possibly requires the binding of transcriptional factor Sp1 to four G-C boxes located -593 to -618 base pairs upstream of the ATG translation initiation codon.

Enhancement of expression of exogenous genes by 2-aminopurine. Regulation at the post-transcriptional level.

In human and mouse cell lines, expression of exogenous genes was enhanced by treatment with 2-aminopurine (2-AP). Chloramphenicol acetyltransferase (CAT) and neomycin phosphotransferase activities were increased by up to 50-fold upon 2-AP treatment of cells permanently transfected with genes encoding these enzymes. Neomycin phosphotransferase activity was also increased by this treatment in cells infected with a retroviral vector carrying the neomycin phosphotransferase gene. 2-AP-mediated increase in CAT activity was observed in various cell lines which had been permanently transfected with chimeric CAT genes containing transcriptional regulatory elements of the interferon-inducible genes 6-16 and 561, SV40 early genes, mouse mammary tumor viral gene, or metallothionein II gene. The increase in the cellular CAT enzymatic activity was due to an elevated level of CAT protein. The 2-AP-mediated enhancement of CAT expression was operative at the translational level; the rate of transcription of CAT mRNA or its steady-state level was affected only marginally. The translational up-regulation by 2-AP was restricted to the genes introduced from outside; there was no gross change in the rate of synthesis of other cellular proteins.

In vivo and in vitro gene transfer to mammalian somatic cells by particle bombardment.

Chimeric chloramphenicol acetyltransferase and beta-galactosidase marker genes were coated onto fine gold particles and used to bombard a variety of mammalian tissues and cells. Transient expression of the genes was obtained in liver, skin, and muscle tissues of rat and mouse bombarded in vivo. Similar results were obtained with freshly isolated ductal segments of rat and human mammary glands and primary cultures derived from these explants. Gene transfer and transient expression were also observed in eight human cell culture lines, including cells of epithelial, endothelial, fibroblast, and lymphocyte origin. Using CHO and MCF-7 cell cultures as models, we obtained stable gene transfer at frequencies of 1.7 x 10(-3) and 6 x 10(-4), respectively. The particle bombardment technology thus provides a useful means to transfer foreign genes into a variety of mammalian somatic cell systems. The method is applicable to tissues in vivo as well as to isolated cells in culture and has proven effective with all cell or tissue types tested thus far. This technology may therefore prove to be applicable in various aspects of gene therapy.

Promoter region of the human CRE-BP1 gene encoding the transcriptional regulator binding to the cyclic AMP response element.

Genomic clones containing the 5'-terminal portion of the human CRE-BP1 gene that encodes transcriptional regulator binding to the cyclic AMP response element (CRE) were isolated. Multiple transcriptional start sites in the promoter region were identified by nuclease S1 mapping and primer extension analysis. By DNase I footprinting with use of purified transcription factor Sp1 and nuclear extracts prepared from HeLa cells, 11 Sp1-binding sites, two CCAAT sequences, two CREs, and three unknown factor recognition elements were found. Transfection of chimeric chloramphenicol acetyltransferase plasmids containing various deletions of the promoter into CV-1 cells indicated that the region between nucleotides -50 and 90, which contained three Sp1-binding sites and one CRE, was sufficient for basal promoter activity. These results suggest that multiple sequence-specific DNA-binding proteins may control the expression of the CRE-BP1 gene, although Sp1 seems to be important for the basal promoter activity.

Molecular and Functional Analysis of the Muscle-Specific Promoter Region of the Duchenne Muscular Dystrophy Gene

Duchenne muscular dystrophy (DMD) gene transcripts are most abundant in normal skeletal and cardiac muscle and accumulate as normal myoblasts differentiate into multinucleated myotubes. In this report we describe our initial studies aimed at defining the cis-acting sequences and trans-acting factors involved in the myogenic regulation of DMD gene transcription. A cosmid clone containing the first exon of the DMD gene has been isolated, and sequences lying upstream of exon 1 were analyzed for homologies to other muscle-specific gene promoters and for their ability to direct muscle-specific transcription of chimeric chloramphenicol acetyltransferase (CAT) gene constructs. The results indicate that the transcriptional start site for this gene lies 37 base pairs (bp) upstream of the 5' end of the published cDNA sequence and that 850 bp of upstream sequence can direct CAT gene expression in a muscle-specific manner. Sequence analysis indicates that in addition to an ATA and GC box, this region contains domains that have been implicated in the regulation of other muscle-specific genes: a CArG box at -91 bp; myocyte-specific enhancer-binding nuclear factor 1 binding site homologies at -58, -535, and -583 bp; and a muscle-CAAT consensus sequence at -394 bp relative to the cap site. Our observation that only 149 bp of upstream sequence is required for muscle-specific expression of a chimeric CAT gene construct further implicates the CArG and myocyte-specific enhancer-binding nuclear factor 1 binding homologies as important domains in the regulation of this gene. On the other hand, the unique profile of myogenic cell line-specific induction displayed by our DMD promoter-CAT gene constructs suggests that other as yet undefined cis-acting sequences and/or trans-acting factors may also be involved.