DEFECTIVE ALLEELS OF GENES PARTICIPATING IN METABOLIC WAYS, AND METHODS FOR THEIR IDENTIFICATION AND USE

摘要

1. A method of screening in vivo for defective alleles encoding enzymes for genes that can be restored by introducing a cofactor, including:! i) introducing into the yeast cells a test allele of an enzyme-encoding gene, the yeast cells containing the first mutation in the first gene that is functionally homologous to the enzyme-encoding gene, and the second mutation in the second gene or group of genes, making the yeast cells dependent on the addition of the cofactor necessary for functioning an enzyme, the first mutation altering the measurable characteristic of yeast cells associated with the function of the first gene; ! ii) adding a cofactor to the culture medium; and! iii) detecting less recovery of the measurable characteristic in the presence of the test allele than in the presence of a wild-type enzyme, thereby revealing incomplete complementation of the first gene mutation with the test allele and identifying the test allele as a defective allele. ! 2. The method according to claim 1, further comprising titrating the amount of added cofactor to determine whether the defective allele is sensitive to the cofactor. ! 3. The method according to claim 1, in which the yeast is diploid. ! 4. The method according to claim 1, wherein the diploid yeast is heterozygous for the test allele of the gene encoding the enzyme. ! 5. The method according to claim 1, in which the first gene is met13, the second gene is fol3. the cofactor is folate, a measurable characteristic is growth, and the enzyme coding gene is selected from the group consisting of MTHFR, MAT1A, MAT2A, GART, MTHFS and ATIC. ! 6. The method according to claim 1, in which the first gene is cys3, the second group of genes is gear