Isolation of the Promoter for Type I Hexokinase from Rat

A genomic clone containing sequence identical to the 5′ region of the cDNA for rat Type I hexokinase was isolated from a λ Charon 4A library. A 5.4-kbEcoRI fragment from this clone, containing the matching sequence, was sequenced in its entirety. Rapid amplification of 5′ cDNA ends (5′ RACE), reverse transcription polymerase chain reaction, and ribonuclease protection experiments were consistent with the existence of multiple transcriptional start sites clustered in three regions approximately 460, 300, and 100 nucleotides upstream from the translational start codon. Together with results of previous work, the 5′ untranslated sequence defined in the present study accounts for the 4.3-kb mRNA for Type I hexokinase seen on Northern blots. Fragments from the 5′ flanking region were cloned into a reporter vector containing the luciferase coding region. Based on transfection experiments with both PC12 and H9c2 cells, promoter activity was associated with a region lying between nucleotide positions −742 and −516 (with A of the ATG codon at the translational start site defined as +1). The promoter region lacks a TATA sequence and, together with the transcriptional start sites, is located within a GC rich segment (a “CpG island”) approximately 1 kb in length. These characteristics have previously been associated with the promoter and transcriptional start sites of genes for “housekeeping enzymes.”