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)

We have constructed a Xenopus laevis oocyte expression vector, pOEV, which allows cloned DNA to be transcribed and translated directly in the oocyte. Since proteins translated in oocytes are post-translationally modified according to conserved eukaryotic signals, these cells offer a convenient system for performing structural and functional analyses of cloned genes. pOEV can be used for direct analysis of proteins encoded by cloned cDNAs without preparing mRNA in vitro, simplifying existing protocols for translating proteins in oocytes with a very high translational yield. Transcription of the vector in oocytes is driven by the promoter for the TFIIIA gene, which can generate 1-2 ng (per oocyte within 2 days) of stable mRNA template for translation. The vector also contains SP6 and T7 promoters for in vitro transcription to make mRNA and hybridization probes. DNA clones encoding chloramphenicol acetyltransferase (CAT) were injected into oocyte germinal vesicles and CAT protein accumulated in the cell over a 2- to 4-day period. We found that the concentration of DNA injected affected protein yields; surprisingly relatively low concentrations in the range 25-50 pg DNA per oocyte gave maximum yields of CAT protein. When as little as 5 pg of pOEV DNA is injected we typically expressed 40 fmol of CAT protein per oocyte, after 4-day incubations. In addition, we have shown that this system is amenable to the expression of nuclear and membrane proteins.
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PMID:pOEV: a Xenopus oocyte protein expression vector. 169 47

The NGFI-A gene encodes a "zinc-finger" protein that is rapidly induced by nerve growth factor (NGF) in PC12 rat pheochromocytoma cells. The complete exon/intron organization and nucleotide sequence of the rat NGFI-A gene have been determined. The gene spans 3789 nucleotides (nt) and is interrupted by a single intron at nt 588. All three zinc-finger DNA-binding domains are contiguously coded for within the 3' exon; this is in contrast to the structure described by others for the Xenopus laevis transcription factor TFIIIA gene. To analyze the transcription of this gene, we have determined the transcription start site and nucleotide sequence of the 5' flanking region. Transfection of PC12 cells with a fragment from the 5' flanking region linked to the chloramphenicol acetyltransferase (CAT) gene revealed that it contains an element which imparts an NGF-inducible phenotype to the normally silent CAT gene. Several regions with homologies to recognizable sequence elements are present in this fragment, including a TATA box at nt -27, serum response elements at nt -84, -106, -370, and -408, a cAMP-responsive element at nt -140, and a transcription factor Sp1-binding site at nt -286. These results establish the genomic structure of this mammalian multifinger protein and demonstrate that an NGF-responsive element lies upstream of the NGFI-A gene.
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PMID:Structure of the NGFI-A gene and detection of upstream sequences responsible for its transcriptional induction by nerve growth factor. 249 4