Work

Construction and expression of mouse thymidylate synthase minigenes.

Year: 1988

Type: article

Abstract: Mouse thymidylate synthase minigenes that lack introns were constructed by ligating restriction fragments containing 4.5, 1.0, or 0.25 kilobase pairs (kb) of 5'-flanking DNA of the normal thymidylate synthase gene and as little as 0.25 kb of 3'-flanking DNA to full-length thymidylate synthase cDNA. All three minigenes were expressed at approximately the same levels following transfection into hamster V79 cells that were deficient in thymidylate synthase. S1 nuclease protection assays revealed that the multiple 5' and 3' termini of thymidylate synthase mRNA in cells transfected with these minigenes were at the same positions as those of the normal mRNA in mouse cells. Deletion analysis of the promoter region revealed that minigenes extending to position -150 nucleotides (relative to the AUG codon) were expressed at approximately the same level as those extending to -1 kb. However, minigenes extending to -53 nucleotides were inactive. To determine if the minigenes were capable of being regulated in a cell cycle-dependent manner, thymidylate synthase gene expression was measured in hamster cells that were stably transfected with the largest minigene and synchronized by serum-stimulation. Thymidylate synthase enzyme level and mRNA content increased 3-5-fold as cells progressed from G1 through S phase.

Source: Journal of Biological Chemistry

DOI: 10.1016/s0021-9258(19)57360-4

Authors: J W DeWille, C H Jenh, T Deng, C J Harendza, L F Johnson

Institution: The Ohio State University

Language: English

Topic: Biochemical and Molecular Research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: hybrid

Estimated APC paid: $3,430

Funders: National Cancer Institute, National Institute of General Medical Sciences