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)

Minus-strand RNA is the first RNA species made by plus-strand RNA viruses, such as mouse hepatitis virus (MHV), and serves as a template for subsequent RNA replication and transcription. The regulation of minus-strand RNA synthesis has been difficult to study because of the paucity of minus-strand RNA. We have optimized a ribonuclease (RNase) protection assay which enabled the detection of minus-strand RNA synthesis from nonreplicating RNAs, thus clearly separating minus-strand from plus-strand RNA synthesis. We used an MHV defective interfering (DI) RNA containing a chloramphenicol acetyltransferase gene as a reporter to determine the cis-acting signal for MHV minus-strand RNA synthesis. It was found that minus-strand RNAs existed in double-stranded RNA form in the cell. By using various deletion clones, we demonstrated that the cis-acting signal for minus-strand RNA synthesis resides in the 55 nucleotides from the 3' end plus poly(A) tail of the MHV genome. This is much shorter than the 436 nucleotides previously reported for the 3'-end replication signal. No specific upstream MHV sequence was required for the initiation of minus-strand RNA synthesis. This finding suggests that the requirement for minus-strand RNA synthesis is much less stringent than that for genomic and subgenomic plus-strand RNA synthesis and that some of the minus-strand RNAs made may not be functional since they may lack the recognition signals for RNA replication or transcription. We further showed that the DI clones which actively transcribed a subgenomic mRNA from an internal intergenic sequence synthesized much less minus-strand RNA than those clones which did not transcribe subgenomic mRNAs, indicating that minus-strand RNA synthesis was inhibited by transcription from an internal promoter of the same DI RNA. This result also suggests that the regulation of the quantities of subgenomic mRNAs is not at the point of minus-strand RNA synthesis but rather at plus-strand RNA synthesis. Furthermore, the finding that the leader sequence was not required for minus-strand RNA synthesis suggests that the leader RNA regulates mRNA transcription during plus-strand RNA synthesis.
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PMID:Identification of the cis-acting signal for minus-strand RNA synthesis of a murine coronavirus: implications for the role of minus-strand RNA in RNA replication and transcription. 796 4

Two kilobase segments of the 5'-untranslated regions of the human and rabbit butyrylcholinesterase (BCHE) genes were characterized. The sequences shared extensive identity except for a 333-base pair (bp) Alu repeat present only in human BCHE. One single transcription start site was found in both genes with the techniques of primer extension, amplification of the 5'-end of mRNA, and RNase protection. Cap sites in human and rabbit BCHE genes were found in strictly homologous positions. In human BCHE, the transcription start site was found 157 bp upstream of Met-28, the translation start site. Potential regulatory elements in both promoters included one AP1 site and multiple sites for topoisomerase, Oct-1 and PEA-3. Transient expression of BCHE-reporter gene constructs showed that a 194-bp fragment of the 5'-flanking region of human BCHE and a 570-bp fragment of rabbit BCHE were sufficient for promoting chloramphenicol acetyltransferase activity in HeLa cells. No consensus TATA and CAAT boxes were found. However, the sequence around the transcription start site exhibited homology with initiator elements found in other TATA-less promoters in developmentally regulated genes.
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PMID:Promoter and transcription start site of human and rabbit butyrylcholinesterase genes. 806 98

The gene encoding a folate-binding protein (FBP) expressed in human placenta has been cloned by screening a genomic library with the KB cell FBP complementary DNA. This gene, contained in a 10-kilobase EcoRI fragment of this genomic clone, has 5 exons, 4 introns, the AATAA polyadenylation signal in the 3'-untranslated region, and a 5'-flanking sequence which contains the promoter elements, all of which span approximately 5 kilobases. Transcription initiation was mapped by RNase protection to a site 73 base pairs downstream from a G-rich sequence linked to a tandemly repeated GGAAG sequence which is a motif that the ets oncogene encoded GA-binding protein (GABP) transcription factor binds. Gel-shift and supershift mobility assays indicate that the G-rich sequence and the ets motif bind specifically to SP1 and GABP, respectively. These cis regulatory elements in tandem drive expression of the chloramphenicol acetyltransferase reporter gene in transiently transfected mouse 3T3 cells. The location of these elements upstream of transcription initiation in this gene, which lacks an appropriately located TATA box promoter, indicates that this SP1-GA binding region most probably regulates expression of this placental FBP. The gene encoding this placental FBP has been assigned the FBP/PL-1 gene because it is a member of a multigene family that includes a gene encoding a FBP expressed in both KB cells and placenta and its unprocessed pseudogene.
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PMID:Characterization of the gene encoding a folate-binding protein expressed in human placenta. Identification of promoter activity in a G-rich SP1 site linked with the tandemly repeated GGAAG motif for the ets encoded GA-binding protein. 810 41

A family of 16 genes encoding the mouse ribosomal protein S24 was identified, and four members from this family were cloned. A single expressed intron-containing S24 gene (termed mrpS24) and one pseudogene (mrpS24p) were completely sequenced and characterized. The mrpS24 gene has seven exons and six introns spanning over 5.1 x 10(3) nucleotides (nt). The cap site of S24 was mapped to a G residue four nt upstream of a polypyrimidine tract and 15 nt downstream of a TATA-like (TATGA) element. The 5' region (-325 to +33) of the mrpS24 gene has a functional promoter that was able to express the fused chloramphenicol acetyltransferase (CAT) reporter gene. Two different forms of mouse S24 cDNA clones were previously isolated. Sequence analysis showed that one of these cDNA clones (termed S24a) lacks the entire exon V sequence (18 nt), and the deduced amino acid sequence is missing a C-terminal lysine residue encoded by the other cDNA (S24b). The pseudogene mrpS24p is flanked by an 11-bp direct repeat, and its sequence is almost identical to the S24 cDNA sequence, but it lacks two mini-exons, V and VI (20 nt), as in the cases of the human and rat S24 cDNAs. RT-PCR experiments demonstrated the existence of a third form (S24c) that similarly lacks both of the mini-exons, and suggested that different species of S24 mRNA might arise from alternative splicing of the mini-exons V and VI. Northern blot analysis showed that S24 expression is down- and up-regulated during adipocyte differentiation and in cellular transformation, respectively. RNase protection assays and RT-PCR experiments suggested that these cell-specific changes of S24 mRNA levels are mainly due to fluctuations in S24c mRNA level. Our results provide the first indication that a ribosomal protein gene is regulated by alternative usage of two mini-exons in a cell-specific manner.
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PMID:Molecular characterization of the mouse ribosomal protein S24 multigene family: a uniquely expressed intron-containing gene with cell-specific expression of three alternatively spliced mRNAs. 812 13

Herpes simplex virus type 1 (HSV-1) expresses a unique series of RNA molecules, the latency-associated transcripts or LATs, during latent infection of neuronal tissues. Previous studies by others have described a TATA box-containing latency-active promoter, referred to here as LAP1, located approximately 700 bp upstream of the 5' end of the major 2.0-kb LAT. In this report, transient gene expression assays were employed to identify a second, novel latency-active promoter (LAP2) present within a region downstream of LAP1 and 5' proximal to the major 2.0-kb LAT. In contrast to LAP1, this promoter lacks a TATA box but possesses cis-acting regulatory elements and other features frequently observed within eukaryotic housekeeping gene promoters. Unlike most other HSV promoters, LAP2 was down-regulated by the viral transcriptional activators ICP4 and ICP0. The majority of LAP2-positive regulatory elements were located within sequences from -257 to -58 relative to the 5' end of the 2.0-kb LAT, and the basal promoter mapped within sequences from -14 to +28. RNase protection experiments demonstrated that chimeric LAT-chloramphenicol acetyltransferase transcripts produced in the transient assays initiated at or near the 5' end of the major 2-kb LAT. Tn5 insertional mutagenesis of the ICP4 regulatory gene determined that down-regulation of LAP2 required the ICP4 transactivating domain and targeted the minimal promoter region as the site of action by ICP4. Replicating recombinant viruses containing a LAP2-lacZ reporter gene cassette in an ectopic site (glycoprotein C locus) were shown to be active in mouse trigeminal ganglia. Taken together, these experiments suggest that the LAT region of the HSV-1 genome contains at least two latency-active promoters which may play different roles in expressing the various LATs. Alternatively, these promoters may comprise a larger promoter-regulatory complex which may influence transcription during latency.
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PMID:A novel latency-active promoter is contained within the herpes simplex virus type 1 UL flanking repeats. 813 9

The catalytic reaction of renin, an aspartyl proteinase, with angiotensinogen is the rate-limiting step fo the renin-angiotensin system involved in the maintenance of blood pressure and electrolyte balance in mammals. We have characterized species-specific expression of the hepatic renin gene by RNase protection experiment, primer extension analysis, and promoter assay using an in vitro DNA transfection. RNase protection experiments revealed that the renin gene is expressed in rat liver, but neither in mouse nor in human. Primer extension analysis identified the putative promoter region of the rat renin gene, which contains TATAAAA sequence, a canonical regulatory DNA element. In order to test whether the upstream region of the renin gene with respect to the putative transcription initiation site is a functional promoter, we have examined the ability of the 5'-flanking sequences of the rat renin gene as well as the human and mouse genes to activate expression of a reporter gene containing the bacterial chloramphenicol acetyltransferase (CAT)-coding sequences, by transient transfection assays. In transfected HepG2 cells, a hepatoma cell line, only the rat renin promoter was capable of driving the CAT gene expression. These results suggested that the rat-specific renin gene expression in the liver could be primarily determined by its promoter specificity.
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PMID:Species-specific expression of the hepatic renin gene. 820 34

Caprine arthritis-encephalitis virus (CAEV) is a lentivirus which is closely related by nucleotide sequence and biological properties to visna virus. Sequence analysis of the CAEV genome revealed the presence of a small open reading frame (ORF) which shares amino acid identity with the visna virus tat gene. Using an infectious molecular clone of CAEV the role of the tat ORF in viral replication was examined. Mutations were made in the tat ORF that introduced two in frame stop codons six amino acids downstream of the tat AUG; in addition, a deletion mutant was made that removed most of the tat ORF. Both of these mutants had greatly reduced virus titers (> 1000-fold less than the wild type infectious clone). Co-transfection of a tat expressing plasmid with these viruses containing the tat ORF mutations resulted in higher levels of virus production demonstrating that the effects of both mutants are tat specific. These mutants provide data that the CAEV tat gene is necessary for efficient virus replication. Analysis of the RNA in these transfected cells showed that complementation of the tat gene was in trans and not the result of recombination. Analysis of the gag and rev proteins in the transfected cells demonstrated that these proteins were not detectable in cells transfected with the tat mutants but could be readily detected when the mutations were complemented in trans with a tat expression vector. To test for tat mediated trans-activation a plasmid expressing the CAEV tat ORF was co-transfected with plasmids containing either the CAEV or visna virus LTR driving transcription of the bacterial chloramphenicol acetyltransferase gene (CAT). These experiments indicate that one function of the CAEV tat protein is to trans-activate gene expression from the viral promoter. RNase protection analysis of CAT mRNA from co-transfected cells demonstrated that CAEV Tat trans-activates gene expression by increasing steady-state levels of mRNA.
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PMID:The CAEV tat gene trans-activates the viral LTR and is necessary for efficient viral replication. 821 71

1. In situ hybridization histochemistry has revealed that neuronal nicotinic acetylcholine receptor subunits are widely expressed in unique although overlapping patterns in the central and peripheral nervous systems. In addition, an intricate regulation of expression is apparent during development. This raises the question of the transcription control of these complex distributions. 2. An analysis of the transcription control elements of one nicotinic receptor subunit, alpha 3, which is expressed both in the central and peripheral nervous systems as well as in PC12 cells, is presented here. 3. Overlapping genomic clones containing the rat beta 4, alpha 3, and alpha 5 gene cluster were isolated and a restriction map of this region was constructed. 4. The transcription start sites of the alpha 3 gene were mapped by RNase protection experiments. Surprisingly, there is no consensus TATA box upstream of these sites. This is consistent with the finding of multiple initiation sites. 5. Functional assays were done in PC12 cells using the bacterial chloramphenicol acetyltransferase gene as a reporter gene. Several restriction fragments from the region located 5' and including the transcription start sites were shown to promote transcription of the reporter gene and thus contain at least part of the alpha 3 promoter. In addition, a region further upstream, within the beta 4 transcription unit, appears to reduce transcriptional activity and thus could be a silencer. 6. It is proposed that this putative silencer is a selective force in maintaining the tight linkage of these three genes.
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PMID:Transcription control elements of the rat neuronal nicotinic acetylcholine receptor subunit alpha 3. 825 14

We have cloned the upstream region of the rat phospholipase C-gamma 1 gene and characterized its promoter activity. The 5'-upstream sequence is highly rich in GC, enriched with CpG dinucleotides and lacks a TATA motif. This sequence also contains many putative binding sites for regulatory proteins. Primer extension and RNase protection assay demonstrated a single transcriptional start site located at 103 nucleotides upstream of the ATG start codon. The transcriptional activities of various 5'-deleted fragments, fused with chloramphenicol acetyltransferase gene, were examined after transfection into C2C12 myoblast. Multiple positive and negative regulatory sites were observed.
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PMID:Promoter region of the rat phospholipase C-gamma 1 gene. 839 38

The human PTH-related peptide (PTHrP) gene comprises eight exons spanning more than 15 kilobases of genomic DNA. The gene has a highly complex controlling region, which contains four alternatively spliced, noncoding exons and at least two putative promoters, one 5' of exon 1A (up-stream TATA element) and the other 5' of exon 2 (down-stream TATA element). To define important cis regulatory sequences of this gene, a functional dissection of PTHrP 5'-flanking DNA was initiated, using chimeric PTHrP-chloramphenicol acetyltransferase (CAT) constructs. This analysis was carried out in PTHrP-negative human renal carcinoma cells, so that RNA derived from transfected DNA could be studied without interference from endogenous PTHrP sequences. Of the initial series of constructs prepared, the most active was a 1.1-kilobase BamHI-HindIII PTHrP-CAT plasmid containing 350 basepairs of DNA 5' of exon 1C and extending into exon 3. Analysis of transfection products by RNase protection and primer extension revealed that this region contains a previously unrecognized promoter of the gene. This element is located immediately 5' of exon 1C, is active in transfected cells when cloned in isolation up-stream of the CAT gene, and appears to be functional in a number of cell lines and tissues on the basis of primer extension analysis. Unlike the other two PTHrP gene promoters, this element is GC rich and does not possess canonical TATA or CAAT sequences. The human PTHrP gene is one of a handful of genes that appear to use both TATA and GC-rich promoter elements.
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PMID:Identification and characterization of a GC-rich promoter of the human parathyroid hormone-related peptide gene. 846 40


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