Cell Growth & Differentiation, Vol 6, Issue 4 409-416, Copyright © 1995 by American Association of Cancer Research
Localization of a cycloheximide-sensitivity response element in the human thymidine kinase gene promoter
L Xia, G Liang, WZ Liu, ZA Yuan and KE Lipson
Department of Molecular Genetics and Microbiology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854, USA.
In order to localize the segment of the human thymidine kinase (TK) gene
promoter that mediates sensitivity of TK mRNA expression to the presence of
cycloheximide (CX), a series of promoter truncation mutants was prepared
between the 460-base pair (bp) promoter that was demonstrated previously to
be sensitive to CX and the 83-bp promoter that was demonstrated previously
to be insensitive to CX. TK promoters containing 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 sequence eliminated CX sensitivity.
Electrophoretic mobility shift assays using a probe containing most of this
region (but omitting the SP1 binding site at the 5'-end of the 130-bp
promoter) identified some complexes whose formation was sensitive to the
presence of CX. Comparison of the sequences of oligonucleotides that were
able to compete for formation of mobility shift complexes identified the
sequence GCGGCC as a putative CX-sensitivity response element. Two such
sequences are found between 83 and 130 bp 5' of the TK capsite. Mutation of
the distal sequence attenuated sensitivity of TK mRNA expression to CX,
while mutation of the proximal sequence had minimal effect on CX
sensitivity. Thus, these data have localized a CX-sensitivity response
element to a segment of TK promoter about 120 bp 5' of the capsite that
includes the hexamer GCGGCC.