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)

The thymidylate synthase (TS) gene is a housekeeping gene that is expressed at much higher levels in proliferating cells than in quiescent cells. We have studied the role of the TS 5'-flanking sequences in regulating the level of expression of the mouse TS gene. A variety of chimeric TS minigenes that contain different promoters linked either to the TS coding region (with or without introns) or to the chloramphenicol acetyltransferase (CAT) coding region were constructed. The activities of the minigenes were determined by transfecting them into cultured cells and measuring the levels of mRNA or enzyme derived from the chimeric genes. We found that the mouse TS promoter had about the same strength as the simian virus 40 early promoter but was significantly stronger than the herpes simplex virus thymidine kinase promoter. Stable transfection studies revealed that minigenes consisting of the normal TS promoter (extending to -1 kb), coding region, and polyadenylation signal were regulated normally in response to growth stimulation. When the TS promoter was replaced by the simian virus 40 early promoter or by a TS promoter that retained only 60 nucleotides upstream of the first transcriptional start site, the minigene was expressed constitutively. A minigene consisting of the TS promoter (extending to -1 kb) linked to the CAT coding region was also expressed constitutively. These observations indicate that sequences upstream of the transcriptional start sites of the TS gene are necessary, although not sufficient, for normal growth-regulated expression of the mouse TS gene.
 ...
PMID:The 5'-flanking region of the mouse thymidylate synthase gene is necessary but not sufficient for normal regulation in growth-stimulated cells. 199 Feb 64

Herpesvirus saimiri has been shown to possess a thymidylate synthase (TS) gene that is unusual in its transcriptional regulation. Although TS is believed to be required for viral DNA synthesis, the TS-specific 2.5-kb mRNA was found most abundantly during the late phases of asynchronous virus replication in permissive cultures. To study the kinetics of gene activation, the TS promoter and regulatory sequences were cloned upstream of the chloramphenicol acetyltransferase (CAT) gene. No CAT expression or transcripts were found after transfection of fusion genes into permissive owl monkey kidney (OMK) cells. However, the promoter was strongly activated when CAT plasmids were cotransfected with intact herpesvirus saimiri virion DNA or were transferred to OMK cells that were lytically infected with herpesvirus saimiri or a related herpesvirus, herpesvirus ateles. CAT was expressed at reduced levels in cultures when viral DNA replication was inhibited by phosphonoacetic acid; this indicates that the gene is activated during the delayed-early phase. However, the highest amounts of mRNA were present in the late period of replication. Deletion analyses localized essential response elements for trans activation in the promoter upstream region between nucleotides -311 and -56; they consisted of related tandem repeats and perfect palindromes. A sequence with two overlapping palindromes of 16 and 18 bp was found to be a major target for activation of the herpesvirus saimiri TS promoter. These palindromes did not have any significant homologies with known sequences of herpesviruses or cellular DNA; the 18-bp palindrome had, however, a certain structural similarity with a conserved sequence of the E2-responsive cis sequence that is required for transcription regulation of early papillomavirus genes.
 ...
PMID:trans activation of the thymidylate synthase promoter of herpesvirus saimiri. 217 Jun 74

To identify the essential motifs of the promoter of the human thymidylate synthase (hTS) gene, we constructed a set of deletion mutants that covers the region from -441 to +28 of the hTS gene. (The nucleotide positions are numbered from the first position of the initiation codon of the hTS gene.) From the results of chloramphenicol acetyltransferase (CAT) assay of these mutants, two positive elements for the promoter activity were identified: one contains CACCC box (CCACACCC) that is found in the SV40 enhancer motif and the other contains the sequence that is homologous to the Sp1 binding site in the mouse TS gene. Furthermore, two negative regulatory sequences were identified between the two positive elements and upstream from the CACCC box. Cassette mutations were introduced into these motifs and the function of the motifs was confirmed. From the results of gel mobility shift analysis, we found that three nucleoprotein complexes were formed in the promoter region of the hTS gene. The formation of one of the complexes was competed by the DNA fragment bearing the GC box. The gel mobility shift analyses using the DNA fragments with cassette mutations revealed that the complex was formed on the Sp1 binding site of the hTS gene and the formation of the complex correlated with the promoter activity of the fragments with cassette mutations measured by the CAT assay.
 ...
PMID:Functional structure of the promoter region of the human thymidylate synthase gene and nuclear factors that regulate the expression of the gene. 884 60

To identify the essential motifs of the promoter of the human gene for thymidylate synthase (TS), we constructed a set of deletion mutants from the 5'-terminal region of the human TS gene. From the results of assays of the expression of chloramphenicol acetyltransferase (CAT), we identified two functional elements with positive effects on the promoter activity: a CACCC box (CCACACCC) and an Sp1-binding motif (GAGGCGGA) that was homologous to the Sp1-binding site in the mouse TS gene. In addition, negative regulatory sequences were identified between the two positive elements and in the region upstream of the CACCC box. The results of gel mobility shift analyses suggested that Sp1 binds to the Sp1-binding motif of the human TS gene promoter and that multiple nuclear factors that are related to Sp1 bind to the CACCC box. Furthermore, the binding of Sp1 to mutated Sp1-binding motifs in the promoter region of the human TS gene was correlated with the promoter activity, as measured by the CAT assay. Therefore, the Sp1 motif seems to be a major contributor to the basic promoter activity of the human TS gene, although multiple positive and negative regulatory elements are involved in the regulated expression of this gene.
 ...
PMID:Identification of functional elements in the promoter region of the human gene for thymidylate synthase and nuclear factors that regulate the expression of the gene. 921 79

Neospora caninum is an obligate intracellular Apicomplexa, a phylum where one of the current methods for functional studies relies on molecular genetic tools. For Toxoplasma gondii, the first method described, in 1993, was based on resistance against chloramphenicol. As in T. gondii, we developed a vector constituted of the chloramphenicol acetyltransferase gene (CAT) flanked by the N. caninum dihydrofolate reductase-thymidylate synthase (DHFR-TS) 5' coding sequence flanking region. Five weeks after transfection and under the selection of chloramphenicol the expression of CAT increased compared to the wild type and the resistance was retained for more than one year. Between the stop codon of CAT and the 3' UTR of DHFR, a Lac-Z gene controlled by the N. caninum tubulin 5' coding sequence flanking region was ligated, resulting in a vector with a reporter gene (Ncdhfr-CAT/NcTub-tetO/Lac-Z). The stability was maintained through an episomal pattern for 14 months when the tachyzoites succumbed, which was an unexpected phenomenon compared to T. gondii. Stable parasites expressing the Lac-Z gene allowed the detection of tachyzoites after invasion by enzymatic reaction (CPRG) and were visualised macro- and microscopically by X-Gal precipitation and fluorescence. This work developed the first vector for stable expression of proteins based on chloramphenicol resistance and controlled exclusively by N. caninum promoters.
...
PMID:The chloramphenicol acetyltransferase vector as a tool for stable tagging of Neospora caninum. 2512 50