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)

The promoter activity of an Acanthamoeba polyubiquitin gene was analyzed in its homologous system. A modified calcium phosphate transfection method using a neomycin marker vector was developed to achieve highly efficient transfection of the Acanthamoeba polyubiquitin gene into Acanthamoeba cells. In this transfection procedure, the calcium phosphate-DNA complex was formed gradually in the medium during incubation with cells and precipitated on the cells. The crucial factors for obtaining efficient transfection were the pH (6.95) of the transfection buffer used for the calcium phosphate precipitation and the amount (25 micrograms/96-well tissue culture plate) and form (circular) of transfecting DNA. Under these conditions, Acanthamoeba isolate 1B6 was transfected at an efficiency of about 40% with the constructed vector pOPSBU, a pOP13CAT-based polyubiquitin gene incorporated neomycin resistance vector. Acanthamoeba polyphaga was transfected at an efficiency of about 10% with this vector. Transfection of both Acanthamoeba strains appeared to result in low copy plasmid integration (about two copies per cell are suggested). The chloramphenicol acetyltransferase (CAT) assays showed that the promoter of the Acanthamoeba polyubiquitin gene in the constructed vector was especially strong in A. polyphaga, thus the pOPSBU-Acanthamoeba system may be useful for the construction of cDNA expression libraries, as well as for the expression of cloned genes.
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PMID:Stable transfection of Acanthamoeba. 909 Jan 13

We have isolated and sequenced a 2388 bp polyubiquitin encoding genomic DNA from Acanthamoeba encompassing two complete and one incomplete ubiquitin units. Codon usage frequency shows extreme bias. The deduced amino acid sequences of each unit are identical to each other and the same as that deduced from a previously sequenced Acanthamoeba castellanii cDNA. The upstream region of this gene, which contained some putative regulatory modules, was recovered by PCR (polymerase chain reaction) amplification and subcloning. This upstream fragment was ligated to the CAT (chloramphenicol acetyltransferase) gene in a eukaryotic expression plasmid and successfully applied to the establishment of an Acanthamoeba transient transfection system. Transfection was performed by electroporation and the optimal voltage was 4500 volts/cm at capacitance 25 microF. DEAE-dextran (25 microg/ml) added into the electroporation buffer increased the transfection efficiency by about 45%. The CAT activity was proportional to the amount of DNA transfected and reached the peak level 48 h after transfection. CAT assays showed that the polyubiquitin gene upstream fragment contains a functional promoter which is about 2.5 times as strong as a viral RSV-LTR promoter when driving CAT expression in Acanthamoeba.
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PMID:An Acanthamoeba polyubiquitin gene and application of its promoter to the establishment of a transient transfection system. 911 25

Ubiquitin-conjugating enzymes (E2 or Ubc) play a key role in the post-translational modification of proteins by ubiquitylation. They are encoded by a large family of genes that are closely related to each other. In this paper we present the first complete structural analysis, including the promoter and the chromosomal location, of a member of this family, the mouse Ubcm4 gene. At the genomic level the Ubcm4 gene spans approx. 50kb and is composed of four exons. Only about 1% of the total gene codes for amino acids. The four different Ubcm4 specific RNAs encode the same protein and differ only in the length of the 3' untranslated region. The polyadenylation signals used by the four different RNAs are all within the 3' terminal exon. At the 5' end of the gene, multiple transcriptional start sites were mapped within a region of 25bp. The region proximal to the initiation sites does not contain a TATA box and is not GC-rich. Transient chloramphenicol acetyltransferase assays, however, showed that this region can promote the expression of a reporter gene and that 15bp upstream of the first initiation site were sufficient for basal expression. The Ubcm4 gene was mapped by interspecific backcross analysis to the proximal region of mouse chromosome 16.
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PMID:Structure of the gene encoding the ubiquitin-conjugating enzyme Ubcm4, characterization of its promoter, and chromosomal location. 993 61