Expression Of Chloramphenicol Acetyltransferase Gene Research Articles

Duplicated CArG box domains have positive and mutually dependent regulatory roles in expression of the human alpha-cardiac actin gene.

An upstream region from the transcription initiation site to -177 base pairs (bp) of the human alpha-cardiac actin gene directs the transient expression of a bacterial chloramphenicol acetyltransferase (CAT) gene only in muscle cells (A. Minty and L. Kedes, Mol. Cell. Biol. 6:2125-2136, 1986). We modified this promoter region by additional 5' deletions, linker-scanning mutations, and insertion-deletion mutations and demonstrated that the asymmetrical sequences in and adjacent to two CArG [for CC(A + T rich)6GG] motifs, located at -140 and -100 bp, play an important positive role in transcription. The significant impairment of transcriptional activity that accompanies the disruption of one CArG box region can be restored by either. This demonstrated that these two elements interact in a mutually dependent and similar manner. Furthermore, a DNA fragment that includes the CArG boxes had significant competitive activity for transcription directed by the alpha-cardiac actin promoter in an in vivo competition assay. We conclude that the two sequences around each CArG box may interact with the same class of trans-acting positive factor(s) and that these interactions may mediate muscle-specific expression. Each of the two CArG regions appears to be bound independently by such a positive factor(s), and the regions support high-level transcription in a synergistic manner. The transcriptional activity of this regulatory region is proportional to its distance from a TATA box (at -30 bp) and is strictly orientation dependent relative to the direction of transcription. Therefore this upstream region is not an enhancer but is a tissue-specific regulatory upstream element.

Baculovirus interaction with nontarget organisms: a virus-borne reporter gene is not expressed in two mammalian cell lines.

The safety of baculoviruses with respect to mammalian species was studied by using a genetically engineered recombinant of Autographa californica nuclear polyhedrosis virus. This recombinant contains the chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian-active promoter and expresses substantial levels of CAT activity on infection of permissive and nonpermissive insect cells (L.F. Carbonell, M.J. Klowden, and L.K. Miller, J. Virol. 56:153-160, 1985). Extremely low levels of CAT activity were detected in mouse and human cell lines that were continuously exposed to the A. californica nuclear polyhedrosis virus recombinant. The appearance of CAT was not inhibited by cycloheximide. Isopycnic centrifugation of purified inoculum showed that a low level of CAT activity was associated with the insect-derived viral particles. Thus, the observed CAT activity is carried into the cells with the virus inoculum, and active expression of the baculovirus-borne CAT gene is not observed in either cell line. The inability of the CAT gene to be expressed in these cell lines with this model system provides additional assurance of the safety of insect baculoviruses with respect to mammalian species.

Autoregulation of bacteriophage P2 repressor.

The immunity repressor of bacteriophage P2 regulates the two convergent promoters, Pe and Pc, located in the early control region. Pe is the early promoter which is negatively regulated by the repressor. It was found, by DNase I protection studies, that the P2 repressor enhances the binding of RNA polymerase to Pc. Furthermore, under in vivo conditions the transcription initiated at Pc, measured as chloramphenicol acetyl transferase gene expression, is low in the absence of repressor but is stimulated by low repressor levels. With increasing repressor concentrations transcription from the Pc promoter decreases. Thus, the P2 repressor both negatively and positively regulates its own promoter.

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was approximately 0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector.

Comparative inhibition of chloramphenicol acetyltransferase gene expression by antisense oligonucleotide analogues having alkyl phosphotriester, methylphosphonate and phosphorothioate linkages.

Several classes of oligonucleotide antisense compounds of sequence complementary to the start of the mRNA coding sequence for chloramphenicol acetyl transferase (CAT), including methylphosphonate, alkyltriester, and phosphorothioate analogues of DNA, have been compared to "normal" phosphodiester oligonucleotides for their ability to inhibit expression of plasmid-directed CAT gene activity in CV-1 cells. CAT gene expression was inhibited when transfection with plasmid DNA containing the gene for CAT coupled to simian virus 40 regulatory sequences (pSV2CAT) or the human immunodeficiency virus enhancer (pHIVCAT) was carried out in the presence of 30 microM concentrations of analogue. For the oligo-methylphosphonate analogue, inhibition was dependent on both oligomer concentration and chain length. Analogues with phosphodiester linkages that alternated with either methylphosphonate, ethyl phosphotriester, or isopropyl phosphotriester linkages were less effective inhibitors, in that order. The phosphorothioate analogue was about two-times more potent than the oligo-methylphosphonate, which was in turn approximately twice as potent as the normal oligonucleotide.

Identification of a promoter element located upstream from the hepatitis B virus X gene.

We have analyzed a series of plasmids in which the sequences located upstream from the hepatitis B virus (HBV) X gene were linked to the chloramphenicol acetyl transferase (CAT) gene. Expression of the marker CAT gene in transfected cells clearly demonstrated that sequences preceding the X gene contain an active promoter. RNA mapping by primer extension indicated that the RNA encoded by the X gene promoter initiates at multiple sites spanning nucleotides 1250 to 1350 on the HBV genome. Deletion within the adjacent HBV enhancer element region significantly reduced the activity of the X gene promoter, suggesting that the X gene promoter requires the enhancer element for maximal activity.

Identification of a promoter element located upstream from the hepatitis B virus X gene.

We have analyzed a series of plasmids in which the sequences located upstream from the hepatitis B virus (HBV) X gene were linked to the chloramphenicol acetyl transferase (CAT) gene. Expression of the marker CAT gene in transfected cells clearly demonstrated that sequences preceding the X gene contain an active promoter. RNA mapping by primer extension indicated that the RNA encoded by the X gene promoter initiates at multiple sites spanning nucleotides 1250 to 1350 on the HBV genome. Deletion within the adjacent HBV enhancer element region significantly reduced the activity of the X gene promoter, suggesting that the X gene promoter requires the enhancer element for maximal activity.

The enhancer sequence of human hepatitis B virus can enhance the activity of its surface gene promoter.

The transcriptional enhancer sequence has recently been demonstrated in the hepatitis B viral genome. This enhancer sequence has also been shown to increase the activity of HBcAg gene promoter. Using the chloramphenicol acetyltransferase gene expression system, we demonstrated that the intrinsic promoter activity of HBsAg gene promoter was stronger than that of HBcAg gene promoter in both human hepatoma cell lines, Hep3B and HuH-7. Furthermore, we showed that the HBV enhancer sequence not only stimulated the HBcAg gene promoter activity, but also stimulated the HBsAg gene promoter activity in both Hep3B and HuH-7 cells. The enhancer sequence increased the HBsAg gene promoter activity 20-fold in both Hep3B and HuH-7 cell lines, while the increase of the HBcAg gene promoter was 2- and 8-fold in Hep3B and HuH-7 cells, respectively.

Rapid radiolabel-sparing thin-layer chromatography method for the visual assessment of chloramphenicol acetyltransferase gene expression.

Journal Article Rapid radiolabel-sparing thin-layer chromatography method for the visual assessment of chloramphenicol acetyltransferase gene expression Get access R. Aubin, R. Aubin Molecular Genetics and Carcinogenesis Laboratory, The W.W.Cross Cancer Institute11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar R. Fourney, R. Fourney Molecular Genetics and Carcinogenesis Laboratory, The W.W.Cross Cancer Institute11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar M. Weinfeld, M. Weinfeld Molecular Genetics and Carcinogenesis Laboratory, The W.W.Cross Cancer Institute11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar M.C. Paterson M.C. Paterson Molecular Genetics and Carcinogenesis Laboratory, The W.W.Cross Cancer Institute11560 University Avenue, Edmonton, Alberta T6G 1Z2, Canada Search for other works by this author on: Oxford Academic PubMed Google Scholar Nucleic Acids Research, Volume 15, Issue 23, 10 December 1987, Page 10069, https://doi.org/10.1093/nar/15.23.10069 Published: 10 December 1987 Article history Received: 02 September 1987 Published: 10 December 1987

Spacing between simian virus 40 early transcriptional control sequences is important for regulation of early RNA synthesis and gene expression.

We have analyzed the effect of insertion mutants between the simian virus 40 (SV40) 21-base pair (bp) repeats and the early-early (EE) TATA sequence. Insertion of 4, 42, or 90 bp of DNA at the SV40 NcoI site (map position 37) has been analyzed for its effect on expression of the SV40 early gene and positioning of the RNA 5' ends. Insertion of 4 bp reduced SV40 early promoter-dependent chloramphenicol acetyltransferase (CAT) expression by six- to eightfold. Increasing the size of the insertion to 42 or 90 bp resulted in a further drop in early gene expression to basal levels. At the RNA level, the 4-bp insertion reduced EE RNA synthesis approximately 10-fold. No concomitant increase in late-early (LE) RNA synthesis was observed. Insertion of 42 or 90 bp of DNA resulted in a decrease of EE RNA synthesis and a stimulation of LE RNA synthesis. Deletion of the SV40 72-bp repeats from the insertion mutants demonstrated that some, but not all, of the LE RNA depends upon the presence of these sequences. These studies suggest that the ability of RNA polymerase II to utilize the EE (TATA-directed) transcriptional control sequence requires an interaction with the upstream 21-bp repeats or the 72-bp repeats or both. That LE RNA levels in pJI1-in42 CAT and pJI1-in90 CAT were equivalent to the level of EE RNA in pJI1-CAT, yet the level of CAT gene expression was decreased greater than 10-fold, suggests that LE mRNA is under translational control and probably prefers a 5' initiation codon proximal to that of the CAT gene.

A single regulatory region modulates both cis activation and trans activation of the herpes simplex virus VP5 promoter in transient-expression assays in vivo.

A detailed analysis of the expression of the bacterial chloramphenicol acetyltransferase gene controlled by the herpes simplex virus major capsid protein (VP5) promoter showed that this promoter can be functionally separated into an 80-base core region, which has the minimal information required to serve as a pol II promoter but which is not fully activated by viral superinfection or by cotransfections with plasmids bearing functional alpha (immediate-early) genes, and an approximately 100-base regulatory region upstream of the core, which allowed full induction of VP5 promoter-driven chloramphenicol acetyltransferase activity but which repressed the ability of the VP5 core promoter to be cis activated by the simian virus 40 enhancer. This was in distinct contrast to the situation with the alkaline exonuclease promoter (a model early promoter) and defined the regions of this promoter which can be used to study the interaction between viral promoters and putative regulatory proteins induced by viral infection.

A single species of pX mRNA of human T-cell leukemia virus type I encodes trans-activator p40x and two other phosphoproteins.

Human T-cell leukemia virus type I (HTLV-I) contains the pX sequence which codes for the trans-activator of the long terminal repeat (LTR) and is thus postulated to be associated with leukemogenesis in adult T-cell leukemia. Overlapping open reading frames (ORF) in the pX sequence were recently found to code for p27x-III and p21x-III by ORF III, in addition to p40x coded for by ORF IV. The mechanism of expression of these newly identified proteins and their possible association with trans-activation were studied. On transfection of an expression plasmid that contains a cDNA sequence of the pX mRNA, products from both ORFs III and IV were detected in the cells. The RNA was synthesized in vitro from the cDNA clone by SP6 RNA polymerase and translated in a rabbit reticulocyte lysate. As translation products, two proteins, p27x-III and p21x-III, were detected in addition to p40x. Elimination of the first and second ATG codons in ORF III resulted in loss of the ability to code for p27x-III and p21x-III, respectively, which indicated that the translations from these two ATG codons were independent. A mutant that lacked both ATG codons was fully active in trans-activation of chloramphenicol acetyltransferase gene expression directed by the LTR. These results indicate that a 2.1-kilobase pX mRNA of HTLV-I independently encodes three proteins, p40x, p27x-III, and p21x-III, by different ORFs and that the last two proteins are not involved in trans-activation of the unintegrated LTR.

Enhanced expression of the bacterial chloramphenicol acetyltransferase gene in mouse cells cotransfected with synthetic polynucleotides able to form Z-DNA.

Recent studies have demonstrated that the left-handed, Z-DNA conformation is favored in polymers containing alternating purine/pyrimidine sequences that can exist in vivo and may play a role in gene expression. On the basis of this assumption, we have studied the effect of various cotransfected polynucleotides on the transient expression of the chloramphenicol acetyltransferase (CAT) gene in thymidine kinase-deficient murine L cells. Cotransfections were performed by calcium phosphate coprecipitation of CAT gene plasmids with various polymers, and the CAT enzymatic activity was measured in cell lysates after 48 hr. About 2- to 10-fold stimulation of CAT gene expression was observed when the cells were cotransfected with 10 micrograms (per 10-cm culture dish) of plasmid pSV2cat, which contains simian virus 40 (SV40) promoter and enhancer sequences, and 2-10 micrograms of polymers that can form Z-DNA, such as poly(dG-m5dC) X poly(dG-m5dC) or poly(dG-dC) X poly(dG-dC), as compared to transfection with pSV2cat alone. Further, enhanced CAT gene expression was also observed when cotransfections were performed with these polymers and two other plasmid vectors, one containing the SV40 promoter but no enhancer and the other lacking any SV40 regulatory sequences. However, poly(dA-dC) X poly(dG-dT), which can form Z-DNA, did not induce any stimulation. Similarly, no or very little stimulation was observed after cotransfection of pSV2cat with either poly(dG) X poly(dC) or poly(dA-dT) X poly(dA-dT), which do not adopt the Z conformation. These results suggest that certain polynucleotides may enhance transcription of the CAT gene.

Upstream regions of the human cardiac actin gene that modulate its transcription in muscle cells: presence of an evolutionarily conserved repeated motif.

Transfection into cultured cell lines was used to investigate the transcriptional regulation of the human cardiac actin gene. We first demonstrated that in both human heart and human skeletal muscle, cardiac actin mRNAs initiate at the identical site and contain the same first exon, which is separated from the first coding exon by an intron of 700 base pairs. A region of 485 base pairs upstream from the transcription initiation site of the human cardiac actin gene directs high-level transient expression of the bacterial chloramphenicol acetyltransferase gene in differentiated myotubes of the mouse C2C12 muscle cell line, but not in mouse L fibroblast or rat PC-G2 pheochromocytoma cells. Deletion analysis of this region showed that at least two physically separated sequence elements are involved, a distal one starting between -443 and -395 and a proximal one starting between -177 and -118, and suggested that these sequences interact with positively acting transcriptional factors in muscle cells. When these two sequence elements are inserted separately upstream of a heterologous (simian virus 40) promoter, they do not affect transcription but do give a small (four- to fivefold) stimulation when tested together. Overall, these regulatory regions upstream of the cap site of the human cardiac actin gene show remarkably high sequence conservation with the equivalent regions of the mouse and chick genes. Furthermore, there is an evolutionarily conserved repeated motif that may be important in the transcriptional regulation of actin and other contractile protein genes.

Expression of Hepatitis B Viral Core Region in Mammalian Cells

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

Expression of hepatitis B viral core region in mammalian cells.

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

Two elements in the bovine leukemia virus long terminal repeat that regulate gene expression.

The bovine leukemia virus, like the human T-cell leukemia viruses (HTLV-I and HTLV-II), are unusual biologically in that viral transcripts are not detected in tumors or infected tissues. The bovine leukemia virus long terminal repeat (BLV LTR) functions as a transcriptional promoter only in cell lines productively infected with BLV. Deletion mapping indicated that at least two regions of the LTR, on the 5' and 3' sides of the RNA start site, influenced gene expression. An analysis has now been made of the effects of coupling sequences from these LTR regions to a heterologous core promoter derived from the SV40 early promoter unit. Through the use of the transient expression of the bacterial chloramphenicol acetyltransferase (CAT) gene to monitor transcriptional activity in vivo, two independent, regulatory elements were identified in the BLV LTR. One was present in a fragment of 75 base pairs derived from the U3 region of the LTR and behaved much like other enhancer elements. It may be a major determinant of BLV expression in productively infected cell lines, since it enhanced transcription controlled by the heterologous core promoter only in these cells. The second element was contained in a 250-bp fragment derived from LTR sequences in the R region, located downstream from the RNA start site. Its activation of CAT expression was not dependent on BLV infection and was evident only when the fragment was located immediately downstream from the RNA start site. BLV expression thus appears to be regulated in part by a cell-specific enhancer element upstream from the core promoter and a novel sequence downstream from the RNA initiation site in the viral LTR.

Autoregulation of adenovirus E1A gene expression

We examined E1A gene expression by two evolutionarily divergent human adenoviruses, type 5 (subgroup C) and type 3 (subgroup B). Adenovirus type 3 (Ad3)-infected A549 cells contained much larger amounts of E1A-specific RNA than adenovirus type 5 (Ad5)-infected cells, from very early (3 h) through the late stages (20 h) after infection. The appearance of such abundant Ad3 E1A transcripts was delayed after infection of Ad5 E1A-expressing 293 cells, suggesting a down regulation of the Ad3 E1A gene by Ad5 E1A gene products. In a reciprocal manner, coinfection of A549 cells led to typically early and intense Ad3 E1A transcription and strongly inhibited transcription of the Ad5 E1A gene. Transient expression assays were developed so that the autoregulation of the E1A gene could be studied apart from the more complex background of infected cells. The DNA sequence surrounding the transcription start site of the Ad3 E1A gene was placed 5' to the sequence which encodes the bacterial chloramphenicol acetyltransferase gene. Cotransfection of HeLa cells with Ad3 or Ad5 E1A-expression plasmids increased the expression of the Ad3 E1A promoter-driven chloramphenicol acetyltransferase gene. Taken together, these results suggest dual autoregulatory features of adenovirus E1A gene expression. The positive and negative effects appear to be temporally distinguished under different conditions, both in viral infection and in transient assays with plasmid-cloned genes.

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLSl, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJ S 140 (6.0 kb), from which pC 194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4(3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLSl. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.

Lens-specific expression and developmental regulation of the bacterial chloramphenicol acetyltransferase gene driven by the murine alpha A-crystallin promoter in transgenic mice.

Two lines of transgenic mice with one to two copies of a DNA fragment containing nucleotides -364 to +45 of the murine alpha A-crystallin gene linked to the bacterial chloramphenicol acetyltransferase (CAT) gene expressed the CAT gene only in their eye lenses. Both CAT activity and alpha A-crystallin were first detected in eyes at approximately 12.5 days of embryonic development, suggesting that the alpha A-CAT fusion gene and the endogenous alpha A-crystallin gene are co-regulated during lens development in the transgenic mice. These experiments show that the murine alpha A-crystallin gene contains a short, cis-acting, tissue-specific regulatory sequence at its 5' end that can target the expression of the bacterial CAT gene, and probably foreign eukaryotic genes, specifically to the ocular lens.