Galactokinase deficiency is an autosomal recessive disorder that causes cataracts in humans. Homozygotes are usually born with cataracts; heterozygotes develop cataracts at a later age. Galactokinase has been mapped by a somatic cell hybrid panel to the region 17q21-22 using starch gel protein electrophoresis. The galactokinase gene has not been cloned nor characterized from any mammal. We propose to isolate the gene for galactokinase to investigate its structure and improve genetic management. Plan:Following the final step of purifying galactokinase from human placenta, two bands are detected by silver stain on a SDS gel. Amino acid analysis has shown the bands to have different composition suggesting they are different proteins. 1) Purify protein, electroblot onto PVDF, sequence both protein bands to generate information for designing oligonucleotides to both protein bands. 2) Screen a cDNA library with mixed oligonucleotides to both proteins. Positive clones will be evaluated for the galactokinase gene by hybridizing to a chromosome 17 hybrid. 3) The putative galactokinase clone hybridizing to the chromosome 17 hybrid will be sequenced, further localized by in situ hybridization to metaphase chromosomes, and analyzed for galactokinase activity in an expression vector. 4) DNA from galactokinase deficient fibroblasts will be sequenced and analyzed for mutations. 5) Site directed mutagenesis will be performed on the galactokinase cDNA to reproduce the mutations found in the galactokinase deficient fibroblasts. The cDNA carrying these mutations will then be inserted into an expression vector to determine their effect on enzyme activity. Other mutations will also be created in the cDNA to further enhance our understanding of the types of mutations that affect galactokinase activity.
| Ai, Y; Zheng, Z; O'Brien-Jenkins, A et al. (2000) A mouse model of galactose-induced cataracts. Hum Mol Genet 9:1821-7 |
| Kolosha, V; Anoia, E; de Cespedes, C et al. (2000) Novel mutations in 13 probands with galactokinase deficiency. Hum Mutat 15:447-53 |
| Bergsma, D J; Ai, Y; Skach, W R et al. (1996) Fine structure of the human galactokinase GALK1 gene. Genome Res 6:980-5 |
| Ai, Y; Jenkins, N A; Copeland, N G et al. (1995) Mouse galactokinase: isolation, characterization, and location on chromosome 11. Genome Res 5:53-9 |
| Stambolian, D; Ai, Y; Sidjanin, D et al. (1995) Cloning of the galactokinase cDNA and identification of mutations in two families with cataracts. Nat Genet 10:307-12 |
