Chloramphenicol Acetyltransferase Assay Research Articles

Regulation of the interleukin-1 beta (IL-1 beta) gene by mycobacterial components and lipopolysaccharide is mediated by two nuclear factor-IL6 motifs.

The cytokines interleukin-1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of IL-1 beta gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and TNF-alpha-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the IL-1 beta gene by deletion analysis and chloramphenicol acetyltransferase assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the nuclear factor-IL6 (NF-IL6) binding site. Site-directed mutagenesis demonstrated that the two NF-IL-6 motifs could be independently activated by LAM, LPS, or TNF-alpha and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or TNF-alpha-stimulated THP-1 cells to the NF-IL6 motifs. We conclude that the two NF-IL6 sites mediate induction of IL-1 beta in response to the stimuli LAM, LPS, and TNF-alpha.

In Vitro DNA Methylation Inhibits FMR-1 Promoter

In fragile X syndrome, the FMR-1 gene is changed by a CGG repeat mutation and an abnormal methylation at a CpG-island 5′ to the gene. To elicit if methylation itself inactivates the gene, FMR-1 promoter was defined by deletion mapping and primer extension assay and was analyzed by in vitro methylation. Promoter activity was measured by transient expression and chloramphenicol acetyl transferase assay. Although this promoter contains several HpaII sites, it was not affected by methylation with HpaII methylase. However, the promoter was completely repressed by methylation with M. SssI which methylates all cytosines of CpG dinucleotides. This repression could not be overridden by SV-40 enhancer. This study indicates that methylation could be the direct cause of FMR-1 inactivation in fragile X syndrome.

Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression.

Sequences which control basal human T-cell lymphotropic virus type I (HTLV-I) transcription probably play an important role in initiation and maintenance of virus replication. We have identified and analyzed a 45-nucleotide sequence (downstream regulatory element 1 [DRE 1]) at the boundary of the R/U5 region of the long terminal repeat which is required for HTLV-I basal transcription. The basal promoter strength of constructs that contained deletions in the R/U5 region of the HTLV-I long terminal repeat were analyzed by chloramphenicol acetyltransferase assays following transfection of Jurkat T cells. We consistently observed a 10-fold decrease in basal promoter activity when sequences between +202 to +246 were deleted. By reverse transcriptase polymerase chain reaction RNA analysis, we confirmed that the drop in chloramphenicol acetyltransferase activity was paralleled by a decrease in the level of steady-state RNA. DRE 1 did not affect the level of Tax1 transactivation. Using a gel shift assay, we have purified a highly enriched fraction that could specifically bind DRE 1. This DNA affinity column fraction contained four detectable proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis: p37, p50, p60, and p100. The affinity column fraction stimulated HTLV-I transcription approximately 12-fold in vitro. No effect was observed with the human immunodeficiency virus or adenovirus major late promoters. Following renaturation of the proteins isolated from an SDS-containing gel, p37, but not the other protein fractions, was able to specifically bind to DRE 1.

Transactivation of Cellular Promoters by an Integrated Hepatitis B virus DNA

A new Hepatitis B virus (HBV) DNA integrant clone DA2-6, isolated from a human hepatocellular carcinoma (HCC) genomic library, was tested for its ability to transactivate expression of other genes. DA2-6 consists of 3.7 kb flanking cellular sequences and an integrated 2.8 kb HBV DNA which covers the region of preS, S, and the 3′ truncated X. Using a chloramphenicol acetyltransferase (CAT) assay, a number of cellular and viral promoters were transactivated by DA2-6,and the spectrum of transactivational effect was the same as that by the wild type X gene of the virus. Deletion mutant analyses indicated that the transactivation function of DA2-6 is expressed by the region that encodes a truncated X-cell fusion product.

Structure of the promoter region of the gene encoding cytochrome c oxidase subunit V in Dictyostelium

A cDNA for the nuclear-encoded subunit V of Dictyostelium discoideum cytochrome-c oxidase was used as a probe to screen a genomic library and isolate the complete gene. Primer-extension analysis revealed two transcription start sites located 32 and 39 nucleotides upstream of the translation initiation codon. The chloramphenicol acetyltransferase assay in transient and stable Dictyostelium transformants indicated that the 400-bp dT-rich segment 5' to the transcription start sites retained promoter activity. This region contains an octanucleotide sequence similar to the yeast HAP2/3/4 responsive element.

NF-IL6 represses early gene expression of human papillomavirus type 16 through binding to the noncoding region

The expression of human papillomavirus type 16 (HPV16) early genes, including E6 and E7 transforming genes, is regulated by several cellular factors binding to the noncoding region (NCR), such as the glucocorticoid receptor, NF-I, and AP1, all of which are positive regulators. We demonstrated that the nuclear factor for interleukin 6 expression (NF-IL6) specifically binds to the HPV16 NCR ranging from nucleotides 7007 to 7766 and represses the early gene expression of HPV16. The responsive element in HPV16 NCR was determined within the region ranging from nucleotides 7454 to 7766. In this region, many binding sites for other cellular transactivators, such as NF-I and AP1, have been detected. Interestingly, three of seven binding sites for NF-I and two of two binding sites for AP1 in this region overlap with the putative NF-IL6 binding sites identified by computer analysis. Competition experiments with the oligonucleotides containing such NF-I or AP1 sites indicated that NF-IL6 certainly binds to them. Furthermore, in a chloramphenicol acetyltransferase assay using mutant NF-IL6 expression vectors, the DNA binding domain of NF-IL6 was shown to be necessary for repression, whereas the functional domain was not. These findings indicate that repression may be caused by competition with other transcriptional activators, such as NF-I and AP1. Thus, NF-IL6 may play a significant role in the regulation of viral transcription as a part of the host's resistance to viral infection.

Acidic fibroblast growth factor gene 5' non-coding exon and flanking region from hamster DDT1 cells: identification of the promoter region and transcriptional regulation by testosterone and aFGF protein.

Selected clones of Syrian hamster DDT1-MF2 cells are responsive to testosterone for growth. Heparin binding growth factor 1 (HBGF-1) or acidic fibroblast growth factor (aFGF) can replace testosterone (T) in the stimulation of growth in these cells. This phenomena is correlated with testosterone's ability to elevate aFGF mRNA two- to threefold in DDT1 cells. To better understand the possible mechanisms of regulation of aFGF mRNA by steroids and other growth factors, we isolated the aFGF 5' non-coding exon and its flanking region from a EMBL3 DDT1 genomic library, using a 5' non-coding exon 69 bp DDT1 aFGF cDNA probe. Clones spanning 30 kb of genomic DNA were isolated. After restriction mapping and DNA sequence analysis, the clones were shown to contain all of the 5' non-coding exon included in the cDNA and approximately 10 kb of 5' flanking region. RNase protection and primer extension assays confirmed that the 5' non-coding exon is included in the DDT1 aFGF mRNA and that a major transcription start site is approximately 136 bp upstream of the 5' non-coding splice junction of this exon. The 5' flanking region DNA was inserted into pBLCAT3 reporter gene and transfected into DDT1 cells. Chloramphenicol acetyltransferase (CAT) assays demonstrated that there are promoter elements in the -1645/-392 and -392/+131 regions of the aFGF gene in the context of DDT1 cells. NIH 3T3 cells, on the other hand, show no CAT activity with these aFGF-CAT plasmids. CAT assays also demonstrated that addition of testosterone (T) or aFGF to DDT1 cells increased CAT activity threefold. This activity was mapped to -1645 to -4 bp region of this DDT1 aFGF gene promoter.

A HPLC-based chloramphenicol acetyltransferase assay for assessing hair growth: comparison of the sensitivity of UV and fluorescence detection.

In our attempt to measure hair growth by hair-specific markers, we used transgenic mice to express the chloramphenicol acetyltransferase gene under the control of an ultrahigh sulphur keratin gene promoter. To quantitate expression of the keratin gene, we required a chloramphenicol acetyltransferase assay which could measure enzyme activity in a single follicle and also could be used to assay a large number of samples without loss of sensitivity. We achieved this objective by utilizing a fluorescent substrate for chloramphenicol acetyltransferase. With HPLC-fluorescence detection, this substrate provides a sensitivity of less than 1 x 10(-13) mol, which is 1000 times greater than that achievable with HPLC-UV detection in cultured follicles. Further, the assay was automated to facilitate the analysis of more than 100 samples/day. It should be possible to apply this fluorescent assay to a number of cell or tissue studies.

Structural analysis of the human insulin-like growth factor-II P3 promoter

The expression of insulin-like growth factor-II (IGF-II) has been observed previously in many human cancers. The human IGF-II P3 promoter has been shown by others to give rise to abundant 6.0 kb and 2.2 kb fetal transcripts which are expressed in a variety of both paediatric and adult tumours. In order to determine the mechanism by which the P3 promoter is controlled, the promoter was analysed in cell lines using chloramphenicol acetyltransferase (CAT) assay and DNAase I footprinting techniques. The data indicated that P3 is a complex promoter involving at least nine transcription factor binding sites. Furthermore, high levels of 5-methylcytosine detected in the P3 promoter of HeLa genomic DNA suggest that IGF-II gene expression may also be influenced by DNA methylation.

Nucleotide sequence and regulated expression of the Salmonella fljA gene encoding a repressor of the phase 1 flagellin gene

The nucleotide sequence of Salmonella abortus-equi fljA, which together with the phase 2 flagellin gene constitutes the fljBA operon and encodes the repressor for the phase 1 flagellin gene fliC, was determined. The repressor was predicted to be a basic protein consisting of 179 amino acid residues (M(r) = 20419 Da) encoded by ORFII. This was confirmed by the fact that host fliC is repressed by plasmid-encoded ORFII, which indeed expresses a 20 kDa product as determined by urea SDS-polyacrylamide gel electrophoresis. An amino acid sequence capable of forming a helix-turn-helix type of structure was predicted in the C-terminal region of FljA. A rho-independent intercistronic terminator was detected between fljB and fljA. Chloramphenicol acetyltransferase (CAT) assays of fusions indicated that the terminator is capable of reducing expression of fljA to the level of a few percent, relative to fljB in broth cultures and to 1% in M9 glycerol cultures.

Differential activity of the RVF enhancer element in the decreased expression of the neu oncogene in NR-6 cells versus parental Swiss Webster 3T3 cells.

The rat neu oncogene encodes a growth factor receptor-like glycoprotein, termed p185, that shares structural similarity with epidermal growth factor receptor (EGFR), particularly in the tyrosine-kinase domain. The Swiss Webster 3T3 murine embryo fibroblast (SW3T3) variant NR-6, in contrast to the parental cell line, does not express EGFR mRNA. After transfection of an activated rat neu cosmid clone, we demonstrated in this report that whereas SW3T3 cells readily expressed the exogenous rat p185 protein, NR-6 cells did not express detectable levels of this protein product. By transfection of plasmids containing the chloramphenicol acetyltransferase (CAT) gene driven by the neu promoter and subsequent CAT assays, we also showed that neu gene promoter activity was significantly less in NR-6 cells than in SW3T3 cells and that the neu promoter sequence responsible for this decreased transcription was a previously identified RVF enhancer element (Yan D-H, Hung M-C, Mol Cell Biol 11:1875-1882, 1991). That is to say, the RVF enhancer element of the neu promoter did not function as an enhancer in NR-6 cells. To investigate the mechanism responsible for the inactivation of RVF in NR-6 cells, we used southwestern blot analyses and demonstrated that the 60-kDa RVF polypeptide was present in both NR-6 and SW3T3 cell nuclear extracts. This result indicates that the DNA-binding activity of RVF was similar in these two cell lines; therefore, loss of RVF enhancer activity in NR-6 cells is probably due to inactivation of the trans-activating function and not DNA binding activity of RVF.

Analysis of the human tyrosine hydroxylase promoter-chloramphenicol acetyltransferase chimeric gene expression in transgenic mice

To investigate cis-elements responsible for catecholaminergic (CAnergic) neuron-specific expression of the tyrosine hydroxylase (TH) gene, we produced lines of transgenic mice carrying 5.0-kb, 2.5-kb and 0.2-kb fragments from the 5′-flanking region of the human TH gene fused to a reporter gene, chloramphenicol acetyltransferase (CAT), and designated them as TC 50, TC 25, and TC 02, respectively, and reporter gene expression in transgenic mice was analyzed by CAT assay by immunocytochemistry with anti-CAT antibody. High-level CAT expression was observed in the brain and adrenal gland using the 5.0-kb promoter of the TC 50 mice, but ectopic expression was consistently observed in several somatic tissues, e.g. thymus, colon, and testis. In brain, expression was achieved in CAnergic neurons with the largest construct (5.0 kb), but not with 2.5 kb or 0.2 kb of 5′ flanking sequence. However, TC 50 mice also expressed CAT immunoreactivity in non-CAnergic neurons. In the TC 25 line CAT immunoreactivity was detected only in some non-CAnergic neurons. In the TC 02 line no CAT immunoreactivity was detected in any of the tissues examined. These results indicate that the 5.0-kb DNA fragment of the TH gene upstream region contains activity to express CAT in CAnergic neurons and surprisingly, lacks some regulatory elements attenuating ectopic expression, and that the 2.5-kb and 0.2-kb fragment are not sufficient for the proper expression. We discuss the presence of the tissue-specific regulatory elements in the structure portion of the TH gene and/or 3′-flanking region.

Identification of two silencers flanking an AP-1 enhancer in the vimentin promoter

We have studied the 5′ upstream sequences required for the transcriptional regulation of the hamster gene encoding the intermediate filament protein, vimentin. Although vimentin is regarded as the intermediate filament protein of mesothelial tissue, it is also produced in most cultured cells. The human mammary carcinoma cell line, MCF-7, belongs to the exceptions. It contains no vimentin, and the complete upstream promoter region is inactive in this particular cell line. By using transient transfection of chimeric constructs into MCF-7 and HeLa cells, and subsequent chloramphenicol acetyltransferase assays, we were able to show the presence of two negative control regions flanking a double AP1 enhancer element. Our data indicate that these elements exert their effect irrespective of orientation and position, suggesting that they are silencers. In vitro footprinting assays, gel mobility assays and Southwestern (protein-DNA) blotting revealed the presence of trans-acting factors interacting with both silencer elements. The silencing effect was particularly pronounced in MCF-7 cells, although DNA-binding proteins are present in HeLa cells as well.

Identification of glucocorticoid responsive elements (GREs) at far upstream of rat NPY gene.

The location of three glucocorticoid responsive elements (GREs) in rat neuropeptide Y (NPY) gene was determined by chloramphenicol acetyltransferase (CAT) assay and nucleotide sequencing. We have reported that mRNA content of rat prepro-NPY is increased by 1.7-fold in NG108-15 cells by 1 microM dexamethasone, suggesting the presence of GRE in the gene. To identify the element, the 5'-flanking DNA of 3.3 kilobases (kb) was isolated from rat NPY gene. When chimeric chloramphenicol CAT plasmids containing various deletions of the NPY upstream sequence were transfected into NG108-15 cells, the region between -2.9 and -2.1 kb relative to the cap site was found to potentiate the transcription of CAT gene in the presence of 1 microM dexamethasone. The nucleotide sequencing of this region revealed three GRE consensus sequences at -2.5, -2.2 and -2.1 kb. The results indicate that these elements present in the far upstream region of the NPY gene confer induction by glucocorticoids.

The upstream region of the mouse N- myc gene: Identification of an enhancer element that functions preferentially in neuroblastoma IMR32 cells

The various members of the myc gene family, including c- myc and N- myc, are supposed to play a role in the regulation of cell cycle and proliferation. Whereas c- myc is expressed nearly ubiquitously, the N- myc gene product is found mainly in actively proliferating neural tissues such as early development tissues or in retinoblastomas and neuroblastomas. In this report, the upstream region of mouse N- myc gene was ligated to pSVPCAT, which carries the simian virus 40 (SV40) promoter and bacterial chloramphenicol acetyltransferase (CAT) gene, and transcriptional activities were examined by CAT and S1 protection assays after transfection of the DNAs into human cervical carcinoma HeLa or neuroblastoma IMR32 cells. Several regulatory regions were identified: two promoting regions (−980 to −860 and −279 to +108) and an inhibiting one (−860 to −797). The region spanning positions −980 to −860 increased CAT expression independently of orientation and distance to the SV40 promoter, indicating that the element is a typical enhancer. Moreover, the expression levels from this enhancer were higher in IMR32 cells than in HeLa cells, indicating that action has, if not cell-type specificity, cell-type preference. These findings may provide useful bases for the understanding of the cell-type specific regulation of N- myc expression.

Production of transgenic tilapia ( Oreochromis niloticus) by one-cell-stage microinjection

Novel genes have been successfully introduced into fish of many species, chiefly by microneedle injection into fertilized eggs. Tilapia are important warm water food fishes with short generation times, but previous attempts to introduce transgenes into this species succeeded only with multicell embryo injection, a procedure that is likely to result in subsequent transgene mosaicism and probable failure of germline transmission to an F1 generation. We here report the successful production of transgenic tilapia ( Oreochromis niloticus) by injection of transgenes (mouse metallothionein promoter/rat growth hormone gene) into the one-celled eggs. Slot blotting reveals the presence of transgene copies in many fish, and Southern blotting patterns suggest genomic integration in roughly 20% of fish developing from injected eggs. Most but not all of the transgenic fish appear to be mosaic in that copies are present only in some tissues. In some male fish analysed, this includes sperm. Introduction of DNA constructs in which a fish gene promoter is spliced to a bacterial CAT (chloramphenicol acetyl transferase) gene, together with subsequent CAT assays, were used to test for gene expression in the transgenic fish. A proportion (approximately 30%) gave positive results in these assays.

Structure, Expression, and Regulation of the Major Noncollagenous Matrix Proteins of Bone

The noncollagenous proteins (NCPs) that predominate the bone matrix have recently been the focus of intense investigation because of their potential influence on cell attachment, Ca2+ and hydroxyapatite binding, and the mineralization of bone tissue. With the advent of molecular biology, all of the major NCPs of bone have been cloned and their amino acid sequences completely determined. While each of the proteins has distinct structural properties, some proteins appear to be part of gene families. Examples include the small proteoglycans, decorin and biglycan, as well as the gamma carboxyglutamic acid proteins, such as matrix gla protein and osteocalcin (bone gla protein). Some of the NCPs that are clearly not members of any known gene family still share several common characteristics. One such example of this "convergent evolution" is bone sialoprotein and osteopontin. Both are highly posttranslationally modified glycoproteins that share the cell attachment amino acid sequence RGD (arginine-glycine-aspartic acid), which facilitates the attachment of bone cells in vitro, yet they are clearly not related genetically. Using cDNAs and antisera as probes, the precise temporal localization of NCP expression has been determined, and it has been shown that NCPs are produced in skeletal, and in most cases, nonskeletal tissue as well. This observation implies that the functions of the NCPs are not necessarily limited to bone tissue. Many of the promoters for these genes have been isolated and functional domains determined by a combination of chloramphenicol acetyltransferase assay, gel shift, and footprint analyses. The most extensively studied promoter in the NCP category is osteocalcin, whose sensitivity to 1,25-dihydroxycholecalciferol has been delineated in detail. Future studies on the individual and cooperative activities of the NCPs in bone are likely to involve site-directed mutagenesis of cloned DNA and a combination of in vitro and in vivo functional analyses.

A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA. A series of mutations in the 5' noncoding region of poliovirus type 3 were introduced into FLC/REP, and their effects were monitored by a simple CAT assay. These experiments helped to define further the stem-loop structures in the 5' noncoding region which are essential for RNA replication. The CAT-containing poliovirus replicon could also be packaged into poliovirus capsids provided by helper virus and was stable as a subpopulation of virus particles over at least four passages. The location of the CAT gene in FLC/REP excluded the presence of an encapsidation signal in the region of the poliovirus genome comprising nucleotides 756 to 1805.

Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.

We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.

High-performance capillary electrophoretic analysis of chloramphenicol acetyl transferase activity

This study highlights the potential utility of high-performance capillary electrophoresis (HPCE) for monotoring enzyme activity. Free-zone capillary electrophoresis is used to rapidly and reproducibly analyze the activity of the bacterial enzyme chloramphenicol acetyl transferase (CAT) which converts the substrates acetyl coenzyme A (CoA) and chloramphenicol to acetyl chloramphenicol and CoA. The results of this study indicate that HPCE may be an excellent tool for studying enzyme activities since it has several advantages over standard single parameters assays, most notably, the ability to monitor both loss of substrate and apperance of products simultaneously. Conditions have been identified for optimal separation of the substrate (chloramphenicol) from the products (acetylated derivatives). This presents a unique potential of HPCE for the analysis of enzymatic reactions that may be applied to areas of analytical research presently utilizing enzymatic reactions. One such analytical method is the CAT assay used for analysis of gene promoter activity. In this study, HPCE is shown to yield similar quantitative results with nonradiolabelled substrate in a fraction of the time. HPCE has several advantages over standard techniques including speed of analysis, no need for radiolabelled substrate, small sample volumes, high sensitivity/resolution and excellent quantitative capabilities.