THE MOUSE TRANSKETOLASE (TKT) GENE - CLONING, CHARACTERIZATION, AND FUNCTIONAL PROMOTER ANALYSIS

The transketolase (TKT) gene is expressed 30-50 times more highly in t he mature mouse cornea than in other tissues. Here, we have cloned and characterized the 30- to 40-kb single-copy mouse TKT gene, Sequence a nalysis supports the suggestion that present-day TKT and TKT-like gene s arose from the duplication of a single common ancestral gene. A 6-bp polymorphism is present between different mouse strains in the noncod ing region of exon 2. 5' RACE and primer extension analyses indicated that two regions separated by 630 bp are used as transcription initiat ion sites; both mRNAs appear to use a common initiator ATG codon, The minor distal transcription initiation site, preceded by a TATA sequenc e, is utilized in liver and is followed by an untranslated exon (exon 1), The major proximal transcription initiation site lies within intro n 1, is used in cornea and liver, lacks a TATA sequence, is GC rich, a nd initiates at multiple sites within a 10-bp span, resembling the pro moters of other housekeeping genes. In transfected cornea and lens cel l lines, the -49/+90 fragment fused to the CAT gene acted as a minimal promoter, with higher activity noted for the -510/+91 fragment. TKT m RNA levels increased sixfold in the mouse cornea in vivo within 1-2 da ys of eye opening and were elevated in a lens cell line exposed to H2O 2 or the glutathione-specific oxidizing agent diamide and in whole new born mouse eyes incubated in the presence of light, consistent with mu ltiple consensus stress-inducible control sequences in the TKT promote r regions, Taken together, these observations suggest that oxidative s tress may play a role in the regulation of this gene in the cornea. (C ) 1998 Academic Press.