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 Thymidylate synthase (TS) is a housekeeping enzyme that is responsible for the formation of thymidylic acid in the de novo biosynthetic pathway. The enzyme is present at a much higher level in proliferating cells that are engaged in DNA replication than in quiescent cells (4,26). We have been studying the mechanisms that are responsible for regulating expression of the mouse TS gene. We have cloned and analyzed the sequences of the cDNA (29) and gene (5) for this enzyme. The gene is 12 kb in length and has a 1-kb coding region interrupted by six introns. The G+C-rich promoter region lacks a TATAA box and initiates transcription at multiple sites over a 60-nucleotide region (5, 8). Mouse TS mRNA is highly unusual in that the predominant species lacks a 3' untranslated region (18). The upstream polyadenylation signal is AUUAAA and is located within the coding region (15).The amount of TS mRNA increases at least 20-fold during a serum-induced transition from the quiescent (GO) phase to the S phase of the cell cycle (8,19 (12,13,21,33,37).Transient transfection assays with TS minigenes have shown that normal levels of gene transcription and the normal pattern of transcriptional start sites are observed with TS 5'-flanking regions that retain as few as 15 nucleotides upstream of the first transcriptional initiation site (or 105 nucleotides upstream of the ATG start codon) (7). Introns are also important for efficient expression of the gene; addition of introns 5 and 6 to an intronless minigene increases the level of expression about eightfold (6). Similar stimulatory effects have been observed previously in other genes (2,3,14). However, addition of intron 4 did not lead to an increase in expression, indicating that the stimulatory effect is not universal for all TS introns (6).In this study, we analyzed the role of the 5'-flanking region in regulating mouse TS gene expression. We constructed a series of chimeric minigenes consisting of different promoters linked to the normal TS coding region and polyadenylation signal. The minigenes were transfected into cultured cells, and the level of expression was measured. Our results indicate that sequences upstream of the essential promoter element(s) are required for normal regulation of TS gene expression in growth-stimulated cells. However, the 5'-flanking sequences are not sufficient for normal regulation. MATERIALS AND METHODSConstruction of mmingenes. The structures of the minigenes used in these analyses are summarized in Fig. 1. The construction of pTTT (also named pTSMG2 [9]) and pTI56T (also named p156 [6]) has been described. Both of these minigenes have 1 kb of the normal 5'-flanking region and 0.25 kb of the normal 3'-flanking region of the mouse TS gene 1023
Three Pd-Ag dental alloys for metal-ceramic restorations, W-1 (Ivoclar Vivadent), Rx 91 (Pentron) and Super Star (Heraeus Kulzer), were subjected to isothermal annealing for 0.5 hr periods in a nitrogen atmosphere at temperatures from approximately 400 degrees to 950 degrees C. The annealing behavior was investigated by Vickers hardness measurements (1 kg load) and SEM microstructural observations. The highest Vickers hardness occurred at approximately 700 degrees C for W-1 and 650 degrees C for Rx 91. For Super Star, there were two peaks in hardness at approximately 500 degrees and 650 degrees C. Additional use of light indenting loads (25 g for W-1; 10 g for Rx 91 and Super Star) revealed that hardness variations during annealing for W-1 and Rx 91 were related to the palladium solid solution matrix phase. For Super Star, the lower-temperature peak was controlled by multi-phase regions and the higher-temperature peak by the matrix phase. While microstructural changes due to annealing were evident with the SEM for Rx 91 and Super Star, no correlation was possible for W-1 because of its finer-scale microstructure. Although commercial Pd-Ag alloys have a relatively narrow composition range, their microstructures and annealing behavior can vary because of differences in proportions of secondary elements utilized for porcelain adherence and grain refinement elements, as well as other proprietary strategies employed by the manufacturers.
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.
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