Expression Of Chloramphenicol Acetyltransferase Gene Research Articles

Binding of Tat Protein to TAR Region of Human Immunodeficiency Virus Type 1 Blocks TAR-Mediated Activation of (2′-5′)Oligoadenylate Synthetase

The TAR sequence of the 5' leader of HIV-1 long terminal repeat-directed mRNA was found to be able to bind to and to activate double-stranded RNA-dependent (2'-5')A synthetase. Binding of TAR to the purified synthetase in vitro was abolished by addition of HIV-1 Tat protein, which binds to this sequence with a high affinity. Inhibition of TAR-mediated activation of (2'-5')A synthetase by Tat was prevented in the presence of the Zn2+ and Cd2+ chelators o-phenanthroline and penicillamine, which did not impair TAR-synthetase interaction. Transient expression assays of bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells revealed that the levels of both CAT mRNA and CAT protein decreased after treatment of the cells with interferon, if CAT gene was linked to HIV-1 TAR segment. Cotransfection of the cells with a tat sequence containing plasmid rendered CAT gene expression insensible to the action of interferon.

MULTIPLE DNA ELEMENTS DETERMINE BASAL AND THYROID HORMONE REGULATED EXPRESSION OF THE HUMAN GLYCOPROTEIN α SUBUNIT GENE IN PITUITARY CELLS

Basal and thyroid hormone-regulated expression of constructs containing varying lengths of 5' flanking sequence of the human glycoprotein hormone alpha subunit gene ligated to the reporter gene encoding chloramphenicol acetyl transferase (CAT) were examined in transfected pituitary GH3 cells. CAT gene expression was quantified by measurement of CAT enzyme activity. In addition, an RNase protection assay was used to confirm changes in CAT mRNA levels and to verify regulation of transcription from the authentic alpha gene start site. The pattern of basal alpha promoter activity indicated the presence of inhibitory DNA elements between -98 and -172 and upstream of -846 relative to the transcriptional start site, as well as a stimulatory element between -346 and -846. Each of the transfected constructs exhibited tri-iodothyronine inhibition of alpha promoter activity, suggesting the presence of an inhibitory thyroid hormone response element within 98 bp of the alpha gene transcriptional start site.

Chloramphenicol-induced translational activation of cat messenger RNA in vitro

The expression of the chloramphenicol-inducible chloramphenicol acetyltransferase gene ( cat) of the staphylococcal plasmid pUB112 is regulated at the post-transcriptional level. Previous in vivo analyses suggested that the antibiotic stalls ribosomes that are translating a regulatory leader peptide, and that a stalled ribosome activates the ribosome binding site of the acetyltransferase encoding sequence by opening an attenuating leader mRNA hairpin structure. To test this model, we used a Bacillus subtilis S-30 extract for an in vitro translation system and in vitro synthesized cat in RNAs. We showed that the leader portion of the cat transcript acts as a translational attenuator of cat gene expression in absence of chloramphenicol. The drug stimulates acetyltransferase synthesis by a leader mRNA-dependent activation of translation of the cat message. By using 5′ end-labeled transcripts and employing the endogenous RNase activity of the S-30 extract we demonstrated that this activation is due to an antibiotic-induced stalling of a ribosome on cat leader mRNA.

Cis-acting regulatory elements near the Epstein-Barr virus latent-infection membrane protein transcriptional start site

Epstein-Barr virus (EBV) latent-infection membrane protein (LMP) gene cis-acting regulatory sequences were assayed in human B lymphocytes by using chloramphenicol acetyltransferase (cat) gene expression as a reporter. The activities of progressively longer upstream elements from bases -55 to -2350 were compared. At least two positive cis-activating regulatory components (-155 to -147 and -234 to -205) upstream of the LMP promoter were defined. LMP promoter cat gene constructs were more active in a Burkitt's lymphoma cell line latently infected with the B95 EBV strain than in the same cells latently infected with the P3HR1 EBV strain. Since the P3HR1- and B95-infected cells differ in EBNA-2 and EBNA-LP expression, EBNA-2 or EBNA-LP is a likely transactivator of the LMP promoter. Probable cognate sequences for known transcription factors in the LMP promoter are discussed.

Cis-acting negative regulatory element of prolactin gene.

The prolactin (PRL) gene in clonal strains of rat pituitary tumor cells in culture (GH cells) exhibit several regulatory responses, similar to the ones observed in rat pituitary gland. A comparative analysis of regulation of PRL gene expression in PRL-producing (PRL+) and PRL-nonproducing (PRL-) GH cells was conducted by monitoring the PRL promoter driven transient expression of chloramphenicol acetyltransferase (CAT) gene in GH4C1 (PRL+) and GH12C1 (PRL-) cells. The PRL promoter activity was drastically inhibited only in PRL-nonproducing cells (PRL-) and not in PRL producing cells (PRL+) when a 80-base pair (bp) DNA sequence from 5'-flanking region of PRL gene (located between -330 and -250 bp) was included in the PRL-CAT fusion gene constructs. Furthermore, a DNA/protein interaction involving this 80-bp DNA sequence and a 60-kDa nuclear protein was detected only in PRL- cells but not in PRL+ GH cells. These results suggested that the strain-specific suppression of PRL gene in PRL-, GH12C1 cells was mediated via interaction of a cis-acting negative regulatory element with a negative regulatory trans-acting factor in these cells. The negative regulatory element within the AT-rich 80-bp DNA sequence was mapped immediately adjacent to the site of interaction of trans-activators of PRL gene.

Host-cell reactivation of cisplatin-damaged pRSVcat in a human lymphoid cell line

A method has been developed for studying host-cell reactivation of cisplatin-damaged plasmid DNA in human T lymphocytes. Parameters of electroporation were established for transfection of the shuttle vector pRSV cat into H9 cells, and a rapid single-vial assay was used for measurement of chloramphenicol acetyltransferase (CAT) activity in extracts of transfected cells. pRSVcat was modified with cisplatin to defined levels ranging from 5 to 40 platinum molecules per plasmid, and then transfected into H9 cells. Graded reductions in CAT activity were observed with increasing levels of platination of the plasmid. At 40 platinum molecules per plasmid, CAT activity was reduced to levels of the negative controls. The efficiency of electroporation-mediated transfer of plasmid into H9 cells was not reduced by high levels of cisplatin modification. The level of modification effecting a 63% reduction in CAT gene expression (the B0), was found to be 17 cisplatin molecules per plasmid. This assay system offers a rapid and sensitive method for assessing the capability of human lymphoid cells to reactivate cisplatin-damaged plasmid DNA.

Effect of the PreS1 RNA sequence on the efficiency of the hepatitis B virus PreS2 and S protein translation

The gene coding for hepatitis B virus surface antigen consists of preS1, preS2, and S regions. Two species of mRNAs of this gene are transcribed. The larger species covers all three regions and is translated solely into preS1 protein, whereas the smaller one covers the preS2 and S regions and is translated into preS2 and S proteins. This study examines the influence of the 5' upstream sequence lying in the preS1 region on the synthesis of preS2 and S proteins. For this purpose, several expression plasmids were constructed by inserting various portions of the preS1 region between the retroviral LTR promoter and the preS2/S coding region, and preS2/S protein production was examined in the transfected CHL cells. All the transcripts were initiated in the LTR. A sequence located in the region between 102 and 38 nucleotides upstream from the preS2 initiation codon was found to reduce the production of preS2/S proteins probably at the level of translation. Expression of the heterologous chloramphenicol acetyltransferase gene was similarly inhibited when it was placed downstream of the preS1 -102/-38 sequence.

Rat β-galactoside α2,6-sialyltransferase genomic organization: alternate promoters direct the synthesis of liver and kidney transcripts

The rat beta-galactoside alpha 2,6-sialytransferase gene is differentially utilized by liver and kidney in the generation of mRNAs that predict substantially divergent polypeptides. In order to determine the biosynthetic relationship between these sialyltransferase mRNA isoforms, genomic sequences were isolated and analysed. Five exons that span at least 40 kb of DNA carry the coding information for the liver beta-galactoside alpha 2,6-sialyltransferase protein. An additional exon contains only sequences for the 5'-untranslated leader of the liver mRNA. In contrast, the predominant kidney mRNAs from this gene share only three coding exons that specify the carboxyl terminal 42% of the liver sialyltransferase protein sequence. In addition, these kidney mRNAs contain information from two other exons that comprise the 5' divergent region of these transcripts. Primer extension and S1 nuclease protection analysis demonstrate that the hepatic and kidney specific mRNAs are transcriptionally initiated at different sites within the sialyltransferase gene. While the hepatic sialyltransferase mRNAs are transcribed from the first exon, the kidney transcripts are initiated from a site within the third intron. Genomic regions upstream of both transcriptional initiation sites can regulate expression of the bacterial chloramphenicol acetyltransferase gene in transiently transfected L cells. Together, the data implicate multiple promoters as a principle mechanism in the generation of kidney and liver gene product diversity in sialyltransferase expression.

Regulation of the murine αA-crystallin promoter in transgenic mice

To identify sequences necessary for lens-specific gene expression, lines of transgenic mice were generated which contain murine αA-crystallin promoter sequences [−111 to +46 (α111), −88 to +46 (α88), and −34 to +46 (α34)] fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and CAT expression was analyzed. Mice carrying the α111-CAT or the α88-CAT fusion transgene expressed CAT exclusively in lens, except for one line containing α111-CAT, which expressed low levels of CAT in several nonlenticular tissues. Transcription from these promoters in lens initiated at the same site as the endogenous αA-crystallin promoter. In one line of mice α88-CAT transgene became active in the lens during embryonic development at approximately the same time that the αA-crystallin gene normally begins to be expressed. In contrast, the α34-CAT fusion transgene, containing the TATA box but no sequences further upstream, was inactive in transgenic mice. Our data suggest that 134 bp of sequence (−88 to +46) in the murine αA-crystallin gene is sufficient to provide lens specificity, although we cannot rule out the possibility that other sequences also contribute to promoter function.

Localization of Elements Important for the Wound-Inducible Expression of a Chimeric Potato Proteinase Inhibitor II-CAT Gene in Transgenic Tobacco Plants.

The effect of progressive 5[prime] deletions within a potato proteinase inhibitor II promoter on wound-inducible expression of the chloramphenicol acetyltransferase (CAT) gene in leaves of transgenic tobacco plants was analyzed. After deletion of a region ranging from position -1300 to -700 with respect to the transcription start site, promoter activity was markedly reduced but still wound-inducible. Further deletion of approximately 200 base pairs resulted in a promoter activity that was below the detection limit, proving that the activity of the proteinase inhibitor II promoter is controlled by sequences upstream of position -514. Addition of the enhancer of the 35S promoter of the cauliflower mosaic virus (CaMV) either 5[prime] upstream or 3[prime] downstream of chimeric genes consisting of different proteinase inhibitor II promoter deletions (-700, -514, -210) fused to the CAT gene led to wound-inducible CAT gene expression in a fraction of transgenic plants containing either the "-700" or "-514" promoters, indicating the presence of wound-responsive elements in the promoter-proximal region. A fragment of the proteinase inhibitor II promoter comprising sequences between positions -1300 and -195 is able to confer wound-inducible expression to an inactive CaMV 35S promoter truncated at position -90 in either orientation, proving that this fragment displays wound-specific, enhancer-like properties. In addition, data are presented excluding that the proteinase inhibitor II 3[prime] end is of importance for wound-inducible gene expression.

Transfection of Leishmania enriettii and expression of chloramphenicol acetyltransferase gene.

We report a transient expression transfection system in Leishmania enriettii. A hybrid gene containing an intergenic region of the alpha-tubulin cluster and the bacterial chloramphenicol acetyltransferase (CAT; EC 2.3.1.28) gene is expressed after transfection of L. enriettii with the hybrid plasmid. The expression of the CAT gene is dependent on the presence of sequences from the alpha-tubulin gene. The hybrid gene is also active in Leishmania braziliensis and Leishmania major.

Transient expression of chloramphenicol acetyltransferase (CAT) gene in barley cell cultures and immature embryos through microprojectile bombardment

Transient expression of chloramphenicol acetyl transferase gene has been detected in cultured barley (Hordeum vulgare L. cv. Heartland) cells and freshly isolated immature zygotic embryos (cv. Ellice) following the introduction of the gene by microprojectile bombardment. The DNA expression vector used to introduce the CAT gene, pCaMVI1CN, is a pUC8 derivative and consisted of a CaMV35S promoter, a fragment of alcohol dehydrogenase intron1, a CAT coding region and NOS polyadenylation region. The inclusion of the Adh1 intron1 was essential for the expression of CAT activity in cultured cells as well as immature zygotic embryos. Expression of CAT activity, which was dependent upon the DNA concentration used, could be detected as early as 20 h after bombardment. The results also suggested that the recipient cells have to be in an active state of cell division in order for the introduced gene to be expressed since mature zygotic as well as somatic embryos failed to reveal any gene expression. The effect of other parameters which influence the expression of the introduced gene as well as the potential of this novel technology for cereal transformation are also discussed.

A direct role for C/EBP and the AP-I-binding site in gene expression linked to adipocyte differentiation.

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

A Direct Role for C/EBP and the AP-I-Binding Site in Gene Expression Linked to Adipocyte Differentiation

Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including the gene encoding adipocyte P2 (aP2), an intracellular lipid-binding protein. Using specific deletions and point mutations, we have shown that at least two distinct sequence elements in the aP2 promoter contribute to the expression of the chloramphenicol acetyltransferase gene in chimeric constructions transfected into adipose cells. An AP-I site at -120, shown earlier to bind Jun- and Fos-like proteins, serves as a positive regulator of chloramphenicol acetyltransferase gene expression in adipocytes but is specifically silenced by adjacent upstream sequences in preadipocytes. Sequences upstream of the AP-I site at -140 (termed AE-1) can function as an enhancer in both cell types when linked to a viral promoter but can stimulate expression only in fat cells in the intact aP2 promoter. The AE-1 sequence binds an adipocyte protein identical or very closely related to an enhancer-binding protein (C/EBP) that has been previously implicated in the regulation of several liver-specific genes. A functional role for C/EBP in the regulation of the aP2 gene is indicated by the facts that C/EBP mRNA is induced during adipocyte differentiation and the aP2 promoter is transactivated by cotransfection of a C/EBP expression vector into preadipose cells. These results indicate that sequences that bind C/EBP and the Fos-Jun complex play major roles in the expression of the aP2 gene during adipocyte differentiation and demonstrate that C/EBP can directly regulate cellular gene expression.

Transcriptional regulatory regions of testis-specific PGK2 defined in transgenic mice.

The gene encoding testis-specific phosphoglycerate kinase 2 (PGK; ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) is expressed only in meiotic and haploid male germ cells. Transgenic mice containing an 8-kilobase human genomic PGK2 gene express the human gene in a tissue-specific and developmentally regulated manner. To determine the nature and location of sequences controlling this expression, transgenic mice with various lengths of the human PGK2 5' region fused to the chloramphenicol acetyltransferase (CAT) gene were analyzed for expression. A 323-base-pair region 5' to the coding region was found to contain information essential for both tissue-specific and developmentally regulated expression of the CAT reporter gene. Transgenic mice containing a PGK2/luciferase-coding construct were compared with mice containing an equivalent CAT construct. Luciferase gene expression was also testis-specific and was more sensitive than CAT gene expression, but otherwise regulation of the two reporter genes was similar in the germ cells of transgenic mice. Translation of both PGK2/CAT and PGK2/luciferase fusion genes was seen concurrently with the first detectable transcripts.

Cadmium-enhanced gene expression in suspension-culture cells of tobacco

The effects of various concentrations of cadmium on Nicotiana tabacum L. cv. Xanthi suspension cells were examined. Surprisingly, certain concentrations of Cd (100-150 μM) stimulated growth of cell cultures considerably, whereas all other concentrations were inhibitory. Synthesis of DNA was severly affected in a dose-dependent manner by Cd concentrations of 250 μM and higher. In contrast, RNA and protein synthesis were similarly stimulated by 100 μM Cd, thus indicating that enhancement of RNA synthesis was the primary cause for the observed stimulation of cell culture growth. The transient expression of a chimeric chloramphenicol-acetyltransferase gene was similarly affected by Cd. When the effects of other heavy metals (Cu, Zn, Pb, Co, Mn, Al) on these cellular processes were investigated, only Zn showed a comparable stimulation of RNA and protein synthesis, although a tenfold higher concentration of Zn compared with Cd was required. As Zn and Cd are chemically very similar, these results are discussed in view of the well-known role of Zn in the regulation of transcription.

Factors affecting transient gene expression in electroporated black spruce (Picea mariana) and jack pine (Pinus banksiana) protoplasts

Methods were developed for transient gene expression in protoplasts of black spruce (Picea mariana) and jack pine (Pinus banksiana). Protoplasts were isolated from embryogenic suspension cultures of black spruce and from non-embryogenic suspensions of jack pine. Using electroporation, transient expression of the chloramphenicol acetyltransferase (CAT) gene was assayed and shown to be affected by the cell line used, by voltage, temperature, and by the plasmid concentration and conformation. Increasing the plasmid DNA concentration (0-150μg ml(-1)) resulted in higher levels of transient CAT expression. In jack pine, linearized plasmid gave 2.5 times higher levels of CAT enzyme activity than circular. Optimal voltage varied for each cell line of the two species within the range 200-350 V cm(-1) (960 μF). A heat shock treatment of protoplasts for 5 min at 45 °C resulted in enhanced CAT gene expression for both species.

An Adenosine 3′,5′-Monophosphate-Responsive Deoxyribonucleic Acid Element Confers Forskolin Sensitivity on Gene Expression by Primary Rat Granulosa Cells*

The following studies were conducted with the goal of understanding some of the molecular mechanisms by which cAMP alters granulosa cell function. In this regard, we characterized the expression of messages for the cholesterol side-chain cleavage cytochrome P450 (P450scc) enzyme and a type II cAMP-dependent protein kinase subunit (RII beta) in granulosa cells isolated from small antral follicles and cultured in 1% fetal bovine serum-containing medium. Forskolin (FSK) stimulated cAMP production followed by accumulation of RII beta mRNAs, induction of P450scc mRNA, and, finally, progesterone biosynthesis. The regulation of each mRNA displayed a different sensitivity to actinomycin-D treatment. To determine if the modulation of RII beta or the induction of P450scc could be mediated by the enhancer activity of a cAMP-responsive DNA element (CRE), plasmid DNA containing the CRE and TATA box of the human glycoprotein alpha-subunit (alpha G) gene fused to the chloramphenicol acetyl transferase (CAT) gene was introduced into cultured granulosa or interstitial cells, and the ability of FSK to stimulate the expression of the CAT enzyme was measured. When granulosa cells were isolated from preantral/small antral follicles and maintained for at least 5 days in culture, 5 or 10 microM FSK reversibly stimulated expression of the CAT enzyme within 18 h posttransfection. Under similar conditions, interstitial ovarian cells (prepared from the residual ovarian tissue remaining after granulosa cell isolation) were unable to express the template unless the cells were pretreated for 24 h with FSK before transfection. Thereafter, CAT gene expression by interstitial cells was maintained in a FSK-insensitive manner. To determine if cAMP-dependent transcription of the reporter gene required the same sequences that had been characterized for placenta-derived cells, a truncated plasmid lacking the CRE of the alpha G gene was transfected. Under no condition was expression of the CAT gene observed from a CRE-deficient template. The acute transcriptional activation of a CRE/TATA box-containing gene by cAMP in granulosa cells indicated that transcription-activating proteins interacting with the CRE were either present in these cells or were rapidly synthesized after stimulation. To distinguish between these two possibilities, protein synthesis was transiently inhibited after transfection, before the addition of FSK, by incubating the cells for 2 h with 25 micrograms/ml cycloheximide. Cycloheximide treatment alone did not stimulate transcription from the CRE-containing molecule. Incubation with cycloheximide, followed by treatment with FSK, increased CAT activity 2-fold compared to t

The mechanism of the bidirectional regulation of the rat α-fetoprotein gene by glucocorticoid hormone

pAFP-CAT, a recombinant plasmid containing 5'-flanking sequence from -7 kb to + 7 bp of rat a-fetoprotein (AFP) gene can drive the expression of the bacterial chloramphenicol acetyltransferase gene in McA-RH7777 and McA-RH8994 rat hepatoma cell lines. Dexamethasone treatment suppresses pAFP-CAT expression in McA-RH7777 cells but increases its expression in McA-RH8994 cells, which mimics the dexamethasone responses of the endogenous AFP gene in both cell lines. However, dexamethasone treatment enhanced pMMTV-CAT expression in both cell lines. These data suggest that the effects of dexamethasone on AFP gene expression may be mediated by different trans-acting factors binding to the specific cis-elements of the 5 '-flanking region of the rat AFP gene.

Tumor necrosis factor alpha activates human immunodeficiency virus type 1 through induction of nuclear factor binding to the NF-kappa B sites in the long terminal repeat.

Expression of human immunodeficiency virus type 1 (HIV-1) can be activated in a chronically infected T-cell line (ACH2 cells) by a cytokine, human tumor necrosis factor alpha (TNF-alpha). TNF-alpha treatment of ACH2 cells resulted in an increase in steady-state levels of HIV RNA and HIV transcription. Gel mobility shift assays demonstrated that the transcriptional activation of the HIV long terminal repeat (LTR) by TNF-alpha was associated with the induction of a nuclear factor(s) binding to the NF-kappa B sites in the LTR. Deletion of the NF-kappa B sites from the LTR eliminated activation by TNF-alpha in T cells transfected with plasmids in which the HIV LTR directed the expression of the bacterial chloramphenicol acetyltransferase gene. Thus, TNF-alpha appears to activate HIV RNA and virus production by ACH2 cells through the induction of transcription-activating factors that bind to the NF-kappa B sequences in the HIV LTR.