Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We describe here a strategy for introducing simultaneous, independent gene replacements into the Trypanosoma cruzi chromosome. The goal of this study was to use two linear DNA fragments to simultaneously replace the CalA2 calmodulin and FUS1 ubiquitin-fusion genes with the neomycin resistance (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. One clone (D6), of thirty G418-resistant clones analyzed, carried the desired dual gene replacement. CDNA sequence analysis indicated that the CAT mRNA was accurately trans-spliced using the previously identified FUS1 mini-exon addition site. However, DNA sequence analysis of the intergenic sequence immediately upstream of the neo(r) gene in clone D6 identified a mutation which altered the pattern of trans-splicing of the neo(r) mRNA. Possible effects of this mutation on 3' splice acceptor site selection are discussed.
 ...
PMID:Analyzing expression of the calmodulin and ubiquitin-fusion genes of Trypanosoma cruzi using simultaneous, independent dual gene replacements. 818 27

Many genes in trypanosomes exist as members of multicopy gene families. Due to this fact it is frequently difficult to determine if specific members of a gene family are expressed. We describe here a strategy for simultaneous tandem gene replacement in T. cruzi which leads to the replacement of the gene of interest by a silent reporter gene, the expression of which can be assayed in stable transformants. To determine if the FUS1 gene (one of 5 copies of the ubiquitin-fusion, FUS, gene family) was expressed, stable G418-resistant transformants were isolated in which the tandemly arrayed CUB2.65 and FUS1 genes were precisely replaced by the neomycin phosphotransferase (neo(r)) and chloramphenicol acetyltransferase (CAT) genes, respectively. All stable clones carrying the tandem gene replacements were shown to express the CAT activity indicating that FUS1 is expressed in mid-log epimastigotes. Northern blot analysis of parasites carrying the tandem gene replacements indicated that at least one other member of the FUS gene family is expressed and that there were no apparent polar effects on the expression of genes downstream of the replacement events. These experiments have demonstrated the utility of tandem gene replacements as a means of inserting a nonselected reporter gene into the chromosome, facilitating the molecular genetic analysis of the expression of multicopy gene families.
 ...
PMID:Using simultaneous, tandem gene replacements to study expression of the multicopy ubiquitin-fusion (FUS) gene family of Trypanosoma cruzi. 823 19