Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six cloned 5' long terminal repeat (LTR) and adjoining cellular DNA regions of partially deleted feline endogenous RD-114 proviral loci were linked to the chloramphenicol acetyltransferase (CAT) gene and assayed for their ability to promote transient CAT expression. One endogenous LTR (clone CRL-3) and the LTR from an infectious RD-114 provirus, EX-LTR, were capable of actively expressing the CAT gene. DNA sequence comparison of these LTRs with an inactive endogenous LTR (CR-1) revealed extensive homology in all regions except in the 5' half of U3. The homologous portion contained transcriptional regulatory sequences including CAT, TATA, polyadenylation signal boxes and an octamer enhancer, which is rarely seen in retroviruses. Variations in the 5' half of U3 were primarily due to insertions and deletions. A major difference was in number of copies and integrity of tandemly repeated sequences. EX-LTR contained two pairs of tandem direct repeats, while the two endogenous LTRs contained different deletions of repeated sequences. DNA sequence data also revealed that the primer binding site for RD-114 loci was complementary to a glycine tRNA isotype, the use of which is distinct from any other known retrovirus. An analysis of the steady state RNA levels in T-lymphoid cell lines showed that at least three different incomplete proviral transcripts and their spliced products made up the majority of expressed RD-114 mRNA, and further demonstrated that partially deleted proviral loci have the potential to be transcriptionally vigorous in certain feline cell types.
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PMID:The long terminal repeat of feline endogenous RD-114 retroviral DNAs: analysis of transcription regulatory activity and nucleotide sequence. 283 49

Caprine arthritis-encephalitis virus (CAEV) and visna virus are pathogenic lentiviruses of goats and sheep which share morphologic features and sequence homology with human T-cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immune deficiency syndrome. The nucleotide sequence of the CAEV long terminal repeat (LTR) was determined, and it was found to be 450 base pairs long, with U3, R, and U5 regions of 287, 85, and 78 base pairs, respectively. Portions of the CAEV LTR are closely homologous to analogous regions of visna virus. The CAEV LTR is not significantly homologous with the HTLV-III LTR; however, like HTLV-III, visna virus, and equine infectious anemia virus, CAEV uses tRNA lysine as a primer for reverse transcription. The transcriptional activity of the CAEV and visna virus LTRs was measured by a chloramphenicol acetyltransferase assay, and the activity of the visna virus LTR was generally higher in a variety of uninfected cell types. Infection of cells with visna virus markedly increased gene expression directed by either the CAEV or visna virus LTR, but in contrast, infection of cells with CAEV had little effect on the activity of either LTR. The lack of trans-activation by CAEV, a virus which causes debilitating arthritis and encephalitis in goats, suggests that trans-activation may not be a general property of pathogenic lentiviruses.
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PMID:Nucleotide sequence and transcriptional activity of the caprine arthritis-encephalitis virus long terminal repeat. 302 73

We have used oligonucleotide-directed site-specific mutagenesis to convert serine codon 27 of the Escherichia coli chloramphenicol acetyltransferase (cat) gene to UAG, UAA, and UGA nonsense codons. The mutant cat genes, under transcriptional control of the Rous sarcoma virus long terminal repeat, were then introduced into mammalian cells by DNA transfection along with UAG, UAA, and UGA suppressor tRNA genes derived from a human serine tRNA. Assay for CAT enzymatic activity in extracts from such cells allowed us to detect and quantitate nonsense suppression in monkey CV-1 cells and mouse NIH3T3 cells. Using such an assay, we provide the first direct evidence that an opal suppressor tRNA gene is functional in mammalian cells. The pattern of suppression of the three cat nonsense mutations in bacteria suggests that the serine at position 27 of CAT can be replaced by a wide variety of amino acids without loss of enzymatic activity. Thus, these mutant cat genes should be generally useful for the quantitation of suppressor activity of suppressor tRNA genes introduced into cells and possibly for the detection of naturally occurring nonsense suppressors.
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PMID:Introduction of UAG, UAA, and UGA nonsense mutations at a specific site in the Escherichia coli chloramphenicol acetyltransferase gene: use in measurement of amber, ochre, and opal suppression in mammalian cells. 302 59

We describe a general protocol for controlled gene amplification, which allows conditional expression of high levels of amber suppressor activity in monkey kidney cells, and we demonstrate its use in the genetic analysis of animal viruses by the generation and propagation of the first nonsense mutant of poliovirus. A human amber suppressor tRNASer gene linked to the SV40 origin of replication and a second DNA carrying a temperature-sensitive SV40 large T antigen gene were cotransfected into monkey cells. Cell lines having stably integrated the DNAs were isolated. Shifting the cells from the nonpermissive temperature to a lower permissive temperature caused the amplification of the suppressor tRNA gene, which resulted in suppression efficiencies at amber codons of 50%-70%, as measured by suppression of an amber codon in the E. coli chloramphenicol acetyltransferase gene. A mutant of poliovirus, in which a serine codon in the replicase gene was converted to an amber codon, was efficiently propagated on the suppressor-positive cell lines. The mutant virus reverted to wild-type by a single base change to a serine codon at a frequency of approximately 2.5 x 10(-6), surprisingly low for a RNA genome.
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PMID:An inducible mammalian amber suppressor: propagation of a poliovirus mutant. 303 32

We succeeded in identifying a promoter element within 200 base pairs upstream a transcriptional unit comprising only a 23S rRNA, 5S rRNA and a tRNA(gly) gene in Thermus thermophilus HB8 [1, 2]. This element shows a high degree of homology to the -35 and -10 consensus sequences for promoters described for Escherichia coli [3, 4]. The promoter activity was measured by the induction of the synthesis of functional chloramphenicol acetyltransferase in Escherichia coli. A region located at the transcriptional start, rich in guanosines and cytidines, is very similar in sequence to the one believed to be under stringent control in stable RNA and ribosomal protein genes of Escherichia coli [5]. Employing nuclease S1 protection we were able to determine the in vivo start of transcription, which was identical with the in vitro start using Escherichia coli RNA-polymerase. Furthermore we identified sequences in the region following the origin of transcription, which are homologous to sections in Escherichia coli rrn promoter-leader regions responsible for antitermination. Our finding of a promoter immediately preceding a 23S/5S rRNA operon proves a transcriptional decoupling of the 16S rRNA genes, a situation so far unprecedented among prokaryotes.
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PMID:An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon. 312 27

The 13 nucleotide Xenopus laevis tyrosine tRNA gene intervening sequence was into a human serine suppressor tRNA gene which lacked an intron, by site-directed mutagenesis. Analysis of the products of in vitro transcription in a HeLa cell extract indicates that the intervening sequence is accurately removed to generate a mature sized RNA identical to that obtained from an intron-less gene. Analysis of the transcripts obtained in vitro and in vivo shows that the U in the CUA anticodon sequence is partially modified to psi. Total TRNA isolated from cells infected with recombinant SV40 viruses carrying the mutant tRNA genes is active in suppression of UAG codons in a reticulocyte cell-free system. Cotransfection of COS cells with the mutant tRNA genes and a mutant chloramphenicol acetyltransferase gene containing the termination codon UAG demonstrated that the tRNA functions as a UAG suppressor in vivo. Analysis of 32P-labeled RNA obtained from infected cells showed, however, that cells infected with the intron-containing gene accumulate less mature tRNA than cells infected with the intron-less tRNA genes.
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PMID:Introduction of an intervening sequence into a human serine suppressor tRNA gene: effects on gene expression in vitro and in vivo. 321 44

Hammerhead ribozyme sequences were incorporated into a tyrosine tRNA (tRNA(Tyr)) and compared with nonembedded molecules. To increase the levels of ribozyme and control antisense in vivo, sequences were expressed from an autonomously replicating vector derived from African cassava mosaic geminivirus. In vitro, the nonembedded ribozyme cleaved more target RNA, encoding chloramphenicol acetyltransferase (CAT), than the tRNA(Tyr) ribozyme. In contrast, the tRNA(Tyr) ribozyme was considerably more effective in vivo than either the nonembedded ribozyme or antisense sequences, reducing CAT activity to < 20% of the control level. A target sequence (CM2), mutated to be noncleavable, showed no reduction in CAT activity in the presence of the tRNA(Tyr) ribozyme beyond that for the antisense construct. The reduction in full-length CAT mRNA and the presence of specific cleavage products demonstrated in vivo cleavage of the target mRNA by the tRNA(Tyr) ribozyme. The high titer of tRNA(Tyr) ribozyme was a result of transcription from the RNA polymerase III promoter and led to the high ribozyme/substrate ratio essential for ribozyme efficiency.
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PMID:Effective ribozyme delivery in plant cells. 759 97

The AGG and AGA are the least used arginine codons in E. coli but they are the most preferable ones in eukaryotes. The low expression of some eucaryotic genes (such as human alpha-1 interferon gene) which contain clusters of AGG codons is explained either by the limited pool of the tRNA(AGG) (Varenne and Lazdunski, 1986) or by the competition of these clusters with the Shine-Dalgarno (SD) sequence (Ivanov et al., 1992). The aim of the present study is to demonstrate the in vivo capacity of AGG tandems to bind to bacterial ribosomes. The two tandems of AGG codons (Arg12 Arg13 and Arg163 Arg164) of hIF alpha 1 with their surrounding nucleotides were cloned in a bacterial expression plasmid containing a strong promoter and a reporter gene (chloramphenicol acetyltransferase, CAT) devoid of a ribosome binding site. The results obtained showed that both AGG tandems initiated translation of the CAT mRNA with an efficiency equal to that of the consensus SD sequence and several fold higher than the native SD sequence of the CAT gene.
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PMID:Domains in human interferon alpha-1 gene containing tandems of arginine codons AGG play the role of translational initiators in E. coli. 764 Oct 76

Eukaryotic cellular mRNA is believed to be synthesized exclusively by RNA polymerase II (pol II), whereas pol I produces long rRNAs and pol III produces 5S rRNA, tRNA, and other small RNAs. To determine whether this functional differentiation is obligatory, we examined the translational potential of an artificial pol III transcript. The coding region of the human immunodeficiency virus type 1 tat gene was placed under the control of a strong pol III promoter from the adenovirus type 2 VA RNAI gene. The resultant chimera, pVA-Tat, was transcribed accurately in vivo and in vitro and gave rise to Tat protein, which transactivated a human immunodeficiency virus-driven chloramphenicol acetyltransferase reporter construct in transfected HeLa cells. pol III-specific mutations down-regulated VA-Tat RNA production in vivo and in vitro and dramatically reduced chloramphenicol acetyltransferase transactivation. As expected for a pol III transcript, VA-Tat RNA was not detectably capped at its 5' end or polyadenylated at its 3' end, but, like mRNA, it was associated with polysomes in a salt-stable manner. Mutational analysis of a short open reading frame upstream of the Tat-coding sequence implicates scanning in the initiation of VA-Tat RNA translation despite the absence of a cap. In comparison with tat mRNA generated by pol II, VA-Tat RNA was present on smaller polysomes and was apparently translated less efficiently, which is consistent with a relatively low initiation rate. Evidently, human cells are capable of utilizing pol III transcripts as functional mRNAs, and neither a cap nor a poly(A) tail is essential for translation, although they may be stimulatory. These findings raise the possibility that some cellular mRNAs are made by pol I or pol III.
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PMID:Functional mRNA can be generated by RNA polymerase III. 779 67

Most eukaryotic cells abundantly express polypeptide chain elongation factor-1 alpha (EF-1 alpha), an enzyme which catalyzes the GTP-dependent binding of aminoacyl-tRNA to ribosomes. In this study, a series of deletion and scanning mutations was introduced in the promoter region of human EF-1 alpha chromosomal gene. Mutated promoters were fused to the bacterial chloramphenicol acetyltransferase gene, and their promoter activity was determined in human HeLa cells. These analyses indicated that both the 5'-flanking region and the first intron of the EF-1 alpha gene are essential for its promoter activity. The region responsible in the intron contains several Sp1 and Ap1 elements which seem to have additive effects on its promoter activity. In the 5'-flanking region, two cis-elements (EFP1 and EFP2) which work interdependently were identified. Gel shift assay with EFP1 and EFP2 elements indicated that several nuclear factors bind to EFP1 and EFP2, and one of the three retarded bands with EFP2 could be super-shifted with the anti-Sp1 antibody. These results indicate that Sp1 or its related factor cooperatively enhances the expression of the EF-1 alpha gene in the 5' flanking region.
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PMID:Characterization of the regulatory elements in the promoter of the human elongation factor-1 alpha gene. 796 76


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