Human HKR isozyme: organization of the hexokinase I gene, the erythroid-specific promoter, and transcription initiation site.
We previously described a cDNA for the human HKR isozyme, whose sequence is identical to that of the ubiquitous HKI isozyme, except for a unique 5' end sequence. Screening a human genomic library with a DNA fragment containing an erythroid-specific sequence we found one clone including 5' ends for both HKR and HKI genes. The first HKR exon was located 3 kb 5' of the first HKI exon. These results confirmed that HKR is produced from the HKI gene by alternate promoter and splicing. The HKI gene consisted of 19 exons. All exon-intron boundaries are conserved among the genes for hexokinase and glucokinase. The HKI gene length was estimated at over 67 kb. The initiation site for the HKR was identified by primer extension. Its promoter did not have a canonical TATA box, but an inverted GATA at nt -177 (i.e., 36 nt 5' to the transcription initiation site). In the HKR promoter a DNA fragment spanning nt -275 to nt -107 exhibited erythroid-specific activity. However, this was absent in shorter promoter fragments (nt -206 to -107 or nt -229 to -107). The sequence nt -275 to -229, which appeared critical for the erythroid-specific expression of the HKR gene, contained a consensus motif for Sp-1 and GATA, CCAAT, and GGAA motifs. The electrophoretic mobility shift assay (EMSA) suggested erythroid-specific cooperative protein-protein interaction in this region. Deletion of the GATA sequence as well as reaction with a specific antibody identified GATA-1 as one of the interacting proteins.
Murakami
K
K
Division of Pediatric Hematology, Columbia University College of Physicians and Surgeons, New York 10032, USA.
Kanno
H
H
Miwa
S
S
Piomelli
S
S
D020121:5' Untranslated Regions / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000378:metabolism*; D005091:Exons; D005796:Genes; D006367:HeLa Cells; D006593:Hexokinase / Q000097:blood / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D007527:Isoenzymes / Q000097:blood / Q000737:chemistry / Q000235:genetics; D020014:K562 Cells; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020218:Response Elements; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D020121:5' Untranslated Regions; D007527:Isoenzymes; D006593:Hexokinase