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)

Androgen receptor (AR) plays a key role in cell growth both in the normal prostate and in prostate cancer. Androgen ablation and prolonged antiandrogen therapy can give rise to AR-dependent prostate tumors, which nonetheless can grow in the androgen-deprived milieu. Here we describe the ribozyme approach to selectively degrading the AR mRNA and thereby inhibiting AR function. A trans-acting hammerhead ribozyme was designed to cleave the rat AR mRNA at the position +1827/ 1828, a region predicted to be minimally involved in generating stable secondary structures. Using AR mRNA fragments as substrates, it was established that this ribozyme can specifically cleave the RNA target in a sequence-specific manner. Kinetic experiments determined a Km for the substrate of 77 nM and a kcat/Km value of 1.8 x 10(7) M(-1) x min(-1), suggesting a catalytic efficiency similar to that of protein enzymes such as the relatively nonspecific ribonuclease A and a sequence-specific endonuclease EcoRI. Transient cotransfections of prostate-derived PC3 cells with three plasmids, an AR-inducible chloramphenicol acetyltransferase (CAT) reporter, an AR expression vector, and a ribozyme expression vector, showed that the ribozyme was capable of reducing the functional activity of AR. At an equimolar ratio of the AR expression plasmid to ribozyme expression plasmid, androgen-inducible CAT activity was inhibited 70%. Similar extents of inhibition were also observed at the cellular mRNA level using ribonuclease protection assays, indicating that the ribozyme functioned as an AR mRNA cleaving enzyme in cellulo. Immunocytochemical examination revealed a decline of AR immunoreactivity in ribozyme-transfected cells. In addition, no morphologically detectable cellular abnormalities were associated with ribozyme expression, indicating the absence of deleterious side effects. These results offer a new avenue for the control of AR function and cell growth, especially in the case of androgen-resistant, but AR-dependent, prostate cancer cells.
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PMID:Catalytic cleavage of the androgen receptor messenger RNA and functional inhibition of androgen receptor activity by a hammerhead ribozyme. 977 79

Genomic DNA libraries were screened for the human histidine-rich glycoprotein (HRG) gene and a sequence of 15,499 nucleotides was determined. The gene is composed of 7 exons and 6 introns, and all the exon-intron boundaries match the consensus GT/AG sequence for donor and acceptor splice sites. Each of cystatin-like domains I and II of HRG is encoded by three exons, exons I to III and exons IV to VI, respectively, like those of other members of the cystatin superfamily. The entire C-terminal half of the molecule is encoded by the largest exon, VII. The first 103 nucleotides of the cDNA sequence reported for human HRG [Koide, T., Foster, D., Yoshitake, S. , and Davie, E.W. (1986) Biochemistry 25, 2220-2225] could not be found in the determined gene sequence. A homology search of this sequence against a database showed the complete matching to a part of the yeast mitochondrial DNA encoding 21S ribosomal RNA. Rapid amplification of cDNA 5' ends (5'-RACE) analysis revealed that the cDNA has multiple 5'-ends and that a possible starting point is nucleotide 104 of the reported cDNA sequence. These results suggest that the first 103 nucleotides of the cDNA sequence reported for human HRG originated from yeast mitochondrial DNA and were incidentally incorporated into the HRG cDNA in the process of the construction of a cDNA library. Various fragments obtained on restriction endonuclease digestion of the 5'-noncoding region of the HRG gene were ligated to the chloramphenicol acetyltransferase (CAT) gene and then transfected into HepG2 and 293 cells to analyze the promoter activity. The sequence between -262 and -21 from the putative translation initiation site supported the expression of CAT in HepG2 cells but not in 293 cells, suggesting that this segment promotes the liver-specific transcription of the human HRG gene.
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PMID:Structural characterization of the gene for human histidine-rich glycoprotein, reinvestigation of the 5'-terminal region of cDNA and a search for the liver specific promoter in the gene. 1005 40

Thirty-nine strains of Salmonella typhi, isolated in 1995 from four Districts in Pakistan, Rawalpindi, Islamabad, Kharian and Jehlem, were catalogued and examined. Chromosomal DNA from each isolate was digested with XbaI restriction endonuclease and subjected to pulsed-field gel electrophoresis. Three clonal variants comprising of 17-19 DNA fragments were identified. Antibiotic susceptibility testing identified that 37 of the S. typhi were resistant to chloramphenicol, trimethoprim and ampicillin. These antibiotic resistance genes were found to be located on one of four plasmids belonging to incompatibility group IncHI1 and ranging in size from 150-175 Kb. The genes responsible for this resistance in each case were the chloramphenicol acetyltransferase (CAT) type I, the dihydrofolate reductase (DHFR) type VII and the beta-lactamase TEM-1 respectively.
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PMID:Characterization of multi-drug resistant Salmonella typhi isolated from Pakistan. 1072 24

Abasic (apurinic/apyrimidinic or AP) sites are a frequent type of DNA damage that threatens genetic stability. The predominant mammalian enzyme initiating repair of AP sites is the Ape1 AP endonuclease (also called Apex or Hap1), which also facilitates DNA binding by several transcription factors (Ref1 activity). We found that expression of the APE1 gene was coordinated with the cell cycle in murine NIH3T3 cells: APE1 mRNA levels rose after the G(1)-S transition and peaked approximately 4-fold higher in early to mid-S phase. The increased APE1 mRNA was the result of transcriptional activation rather than increased mRNA stability. Fusions of various APE1 promoter fragments to the chloramphenicol acetyltransferase CAT reporter gene indicated that APE1 expression depends on two transcription factor Sp1 binding sites within the promoter region. Mutation of these sites or of two CCAAT elements within the APE1 promoter, in conjunction with protein binding studies, demonstrated their specific roles. The Sp1 site upstream of the transcription start, together with an adjacent CCAAT element, establishes a protein-DNA complex required for basal transcription of APE1. The Sp1 site downstream of the transcription start was required for the response to cell growth. Because Ape1 is a dual function enzyme, its cell cycle-dependent expression might affect both DNA repair and the activity of various transcription factors as a function of the cell cycle.
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PMID:Key role of a downstream specificity protein 1 site in cell cycle-regulated transcription of the AP endonuclease gene APE1/APEX in NIH3T3 cells. 1155 53

The Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV) alkaline nuclease (AN) associates with the baculovirus single-stranded DNA binding protein LEF-3 and possesses both a 5'-->3' exonuclease and an endonuclease activity. These activities are thought to be involved in DNA recombination and replication. To investigate the role of AN in AcMNPV replication, the lambda Red system was used to replace the an open reading frame with a chloramphenicol acetyltransferase gene (cat) and a bacmid containing the AcMNPV genome in Escherichia coli. The AcMNPV an knockout bacmid (vAcAN-KO/GUS) was unable to propagate in Sf9 cells, although an an-rescued bacmid (vAcAN-KO/GUS-Res) propagated normally. In addition, the mutant did not appear to produce budded virions. These data indicated that an is an essential baculovirus gene. Slot blot and DpnI assays of DNA replication in Sf9 cells transfected with vAcAN-KO/GUS, vAcAN-KO/GUS-Res, and a wild-type bacmid showed that the vAcAN-KO/GUS bacmid was able to replicate to levels similar to those seen with the vAcAN-KO/GUS-Res and wild-type bacmids at early stages posttransfection. However, at later time points DNA did not accumulate to the levels seen with the repaired or wild-type bacmids. Northern analysis of Sf9 cells transfected with bacmid vAcAN-KO/GUS showed that transcription of late and very late genes was lower at later times posttransfection relative to the results seen with wild-type and vAcAN-KO/GUS-Res bacmids. These data suggest that the an gene might be involved in the maturation of viral DNA or packaging of the DNA into virions.
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PMID:Characterization of a baculovirus lacking the alkaline nuclease gene. 1536 32

Escherichia coli cell-free protein synthesis is a highly productive system that can be applied to high throughput expression from polymerase chain reaction (PCR) products in 96-well plates for proteomic studies as well as protein evolution. However, linear DNA instability appears to be a major limitation of the system. We modified the genome of the E. coli strain A19 by removing the endA gene encoding the endonuclease I and replacing the recCBD operon (in which recD encodes the exonuclease V) by the lambda phage recombination system. Using the cell extract from this new strain increased the stability of PCR products amplified from a plasmid containing the cat gene. This resulted in CAT (chloramphenicol acetyltransferase) production from PCR products comparable to that from plasmids (500-600 microg/ml) in a batch reaction. We show that cell-free protein synthesis reactions using PCR products amplified from genomic DNA and extended with the T7 promoter and the T7 terminator give the same high yields of proteins (550 microg/ml) in 96-well plates. With this system, it was possible to rapidly express a range of cytoplasmic and periplasmic proteins.
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PMID:Increasing PCR fragment stability and protein yields in a cell-free system with genetically modified Escherichia coli extracts. 1625 43


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