LOCALIZATION OF A CYCLOHEXIMIDE-SENSITIVITY RESPONSE ELEMENT IN THE HUMAN THYMIDINE KINASE GENE PROMOTER

In order to localize the segment of the human thymidine kinase (TK) ge ne promoter that mediates sensitivity of TK mRNA expression to the pre sence of cycloheximide (CX), a series of promoter truncation mutants w as prepared between the 460-base pair (bp) promoter that was demonstra ted previously to be sensitive to CX and the 83-bp promoter that was d emonstrated previously to be insensitive to CX. TK promoters containin g 370, 300, 160, or 130 bp of 5'-flanking sequence were all sensitive to inhibition by CX. Further truncation to 100 bp of 5'-flanking seque nce eliminated CX sensitivity. Electrophoretic mobility shift assays u sing a probe containing most of this region (but omitting the SP1 bind ing site at the 5'-end of the 130-bp promoter) identified some complex es whose formation was sensitive to the presence of CX. Comparison of the sequences of oligonucleotides that were able to compete for format ion of mobility shift complexes identified the sequence GCGGCC as a pu tative CX-sensitivity response element. Two such sequences are found b etween 83 and 130 bp 5' of the TK capsite. Mutation of the distal sequ ence attenuated sensitivity of TK mRNA expression to CX, while mutatio n of the proximal sequence had minimal effect on CX sensitivity. Thus, these data have localized a CX-sensitivity response element to a segm ent of TK promoter about 120 bp 5' of the capsite that includes the he xamer GCGGCC.