Analysis of the multiple 5′ and 3′ termini of poly(A) + and poly(A)− deficient thymidylate synthase mRNA in growth‐stimulated mouse fibroblasts

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Abstract
Thymidylate synthase (TS) mRNA content increases about 20-fold when growth-stimulated mouse cells progress from the GO/G1 phase into the S phase of the cell cycle. Previous studies, using a cell line in which the TS gene is amplified (LU3-7), indicated that transcriptional initiation as well as polyadenylation of the mRNA occur at several locations in unsynchronized cells. In the present study, we have used S1 nuclease protection assays to analyze the possible significance of the multiple transcriptional initiation and polyadenylation sites. We found that the same pattern of 5′ and 3′ termini were detected with RNA isolated from the overproducing cells as with RNA isolated from the parental mouse 3T6 cell line, demonstrating that the heterogeneous termini are not a consequence of gene amplification. There was no change in the pattern of 5′ or 3′ termini with either cell line during the progression from G1 phase through S phase in serum-stimulated cells. Therefore, the increase in TS mRNA content is not the result of differential utilization of the various transcriptional initiation or polyadenylation sites. Analyses of poly(A)− deficient cytoplasmic TS RNA showed that the 5′ termini were the same as those found in poly(A)+ mRNA. However, the 3′ termini were extremely heterogeneous in length. Although some of the poly(A)− deficient RNA extended beyond the normal site of polyadenylation, most of it was shorter than full-length TS mRNA.