The mouse transketolase (TKT) gene: cloning, characterization, and functional promoter analysis

The transketolase (TKT) gene is expressed 30-50 times more highly in the mature mouse cornea than in other tissues. Here, we have cloned and characterized the 30- to 40-kb single-copy mouse TKT gene. Sequence analysis supports the suggestion that present-day TKT and TKT-like genes arose from the duplication of a single common ancestral gene. A 6-bp polymorphism is present between different mouse strains in the noncoding region of exon 2. 5' RACE and primer extension analyses indicated that two regions separated by 630 bp are used as transcription initiation sites; both mRNAs appear to use a common initiator ATG codon. The minor distal transcription initiation site, preceded by a TATA sequence, is utilized in liver and is followed by an untranslated exon (exon 1). The major proximal transcription initiation site lies within intron 1, is used in cornea and liver, lacks a TATA sequence, is GC rich, and initiates at multiple sites within a 10-bp span, resembling the promoters of other housekeeping genes. In transfected cornea and lens cell 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 mRNA levels increased sixfold in the mouse cornea in vivo within 1-2 days of eye opening and were elevated in a lens cell line exposed to H2O2 or the glutathione-specific oxidizing agent diamide and in whole newborn mouse eyes incubated in the presence of light, consistent with multiple consensus stress-inducible control sequences in the TKT promoter regions. Taken together, these observations suggest that oxidative stress may play a role in the regulation of this gene in the cornea.