Galactokinase is an essential enzyme in the metabolism of galactose. Patients with deficiencies in galactokinase exhibit early‐onset cataracts. We examined the sequence of the human galactokinase gene (GK1) from 13 patients exhibiting galactokinase deficiency and identified 12 novel mutations. One of the mutations occurred in six of the 13 probands examined, and the remaining 11 were unique mutations. Expression of each of the mutant GK1 genes in Xenopus oocytes resulted in very low galactokinase activity levels. These results provide important information regarding the types of GK1 mutations that occur in the human population. Hum Mutat 15:447–453, 2000. © 2000 Wiley‐Liss, Inc.
Defects in the human C, ALKI gene result in galactokinase deficiency and cataract formation. We have isolated this gene and established its structural organization. The gene contains 8 exons and spans -7.3 kb of genomic DNA. The GALK1 promoter was localized and found to have many features in common with other housekeeping genes, including high GC content, several copies of the binding site for the Spl transcription factor, and the absence of TATA-box and CCAAT-box motifs typically present in eukaryotic Pol 11 promoters. Analysis by S'-RACE PCR indicates that the GALKI mRNA is heterogeneous at the 5' terminus, with transcription sites occurring at many locations between 21 and 61 bp upstream of the ATG start site of the coding region. In vitro translation experiments of the GALK1 cDNA indicate that the protein is cytosolic and not associated with the endoplasmic reticulum membrane.Genetic defects of galactose metabolism constitute a class of genetic disorders termed galactosemia. One form of galactosemia in humans is caused by an enzyme deficiency of galactokinase (Segal 1989). Individuals with homozygous galactokinase deficiency are symptomatic in the early infantile period with galactosemia, galactosuria, increased galactitol levels, cataracts, and in a few cases, mental retardation (Segal et al. 1979). Heterozygotes for galactokinase deficiency are prone to presenile cataracts at -20-50 years of age (Stambolian et al. 1986).The isolation and characterization of the galactokinase gene has been completed in Escherichia coli, Saccharomyces, and Streptomyces lividans (Citron and Donelson 1984;Debouck et al. 1985;Adams et al. 1988). Recently the cDNA encoding human galactokinase, termed GALK1, was cloned, characterized functionally, and mapped to chromosome 17q24 . Analysis of the sequence predicted a 1.35-kb mRNA that results in a polypeptide chain of 392 amino acids. This was confirmed by detection of a single 1.35-kb mRNA on a Northern blot. The 3' end of the mRNA was well defined, but the 5' end was not defined completely. Analysis of the predicted protein sequence showed a conserved galactokinase signature sequence as well as two different ATP-binding motifs that are conserved among all the galactokinases. Additionally, two distinct mutations were identified within GALK1 gene-coding sequences of two unrelated families with galactokinase deficiency and cataracts.Definition of the GALK1 gene structure will improve our understanding of the protein function by allowing experimental manipulation of the gene in vitro and in vivo through gene knockouts. It will also facilitate further genetic studies of patients with galactokinase deficiency. In this study we have determined the complete exon-intron structure of the human GALK1 gene and examined expression and processing of the encoded protein. RESULTS AND DISCUSSION Isolation of the Human GALKI GeneSix phage clones were isolated from a human placental genomic library by screening with the fulllength GALK1 cDNA and characterized by restriction mapping followed by Sou...
Galactokinase is an essential enzyme in the metabolism of galactose. Patients with deficiencies in galactokinase exhibit early-onset cataracts. We examined the sequence of the human galactokinase gene (GK1) from 13 patients exhibiting galactokinase deficiency and identified 12 novel mutations. One of the mutations occurred in six of the 13 probands examined, and the remaining 11 were unique mutations. Expression of each of the mutant GK1 genes in Xenopus oocytes resulted in very low galactokinase activity levels. These results provide important information regarding the types of GK1 mutations that occur in the human population.
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