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Nucleic Acids Research, 1992, Vol. 20, No. 15 4021-4025
© 1992

MOLECULAR BIOLOGY

Intron 1 and the 5'-flanking region of the human thymidylate synthase gene as a regulatory determinant of growth-dependent expression

Atsushi Takayanagi, Sumiko Kaneda, Dai Ayusawa1 and Takeshi Seno*

Laboratory of Mutagenesis, National Institute of Genetics and Department of Genetics, The Graduate University of Advanced Studie Mishima 411 1Institute of Applied Microbiology, University of Tokyo Bunkyo-ku, Tokyo 113, Japan

*To whom Correspondence should be addressed

Received April 15, 1992. We have determined the regulatory regions responsible for the growth-dependent expression of the human thymidylate synthase (TS) gene, using a set of minigenes constructed from segments of the human TS gene and the cDNA clone. Each construct was introduced stably Into a TS-negative mutant of rat fibroblast 3Y1 cells. By serum-restricted synchronization of the cloned transformant cells, we found that a minigene with the genomic 5'-flanking region and intron 1 without other introns were sufficient for the normal extent and pattern of S-phase specific expression at the levels of both mRNA and enzymatic activity. In contrast, a TS cDNA clone driven by an SV40-based expression vector showed constitutive expression. Insertion of intron 1 into the cDNA clone In the normal location, or replacement of the viral 5'-promoter region of the cDNA clone by the genomic 5'-flanking sequence converted the constitutive expression to the S-phase dependent one, but only partly, that is, coexistence of the two regions were required for the normal expression. Results obtained by nuclear run-on assay suggested that posttranscriptional controls are also Involved in this regulation in consistent with our previous results with the bona fide human TS gene.


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