Human leukemia cell K562 in culture displayed an absolute requirement for folate, proliferating at optimal rate only when the total intracellular folate concentration was greater than 1.5 uM. This critical intracellular concentration was maintained by extracellular 5-formyltetrahydrofolic acid or 5-methyltetrahydrofolic acid at a 100-fold lower concentration than folic acid. Growth rate of folate-deficient cells was proportional to intracellular concentration of fully-reduced, non-methylated folate but not to intracellular 5-methyltetrahydrofolate concentration. Growth of folate-deficient cells was stimulated by inosine but not by thymidine, suggesting that the primary cause of growth restriction in these cells was purine deficiency. Serine stimulated, and glycine inhibited, growth of K562 cells, likely by modulating the supply of one-carbon units for folate metabolism. Folate-deficient cells were larger in size than folate-replete cells, but megaloblastic morhology was not observed. DNA content of folate-deficient cells was predominantly characteristic of cells in S or G2/M phases of the cell cycle.

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Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a methyl donor for the re-methylation of homocysteine to methionine. A thermolabile variant of this enzyme, present in approximately 35% of alleles in the North American population, has been associated with cardiovascular disease, neural tube defects, and colon cancer. A cDNA of 2.2kb for human MTHFR has been expressed and results in an active enzyme, but the cDNA and genomic sequences 5' to the ATG start site have not been adequately investigated. The characterization of the 5' region of the human MTHFR gene is reported here. Four additional 5' exonic sequences were localized to a 4kb genomic fragment. The original exon 1 extends directly upstream into a 5' UTR. Three other 5' exons (two with open reading frames) are alternatively spliced into a common splice acceptor site, generating cDNAs with 4 possible 5' ends. The N-terminal peptide sequence of the porcine MTHFR has not been identified in the human sequence suggesting that the missing human coding sequence might be localized further upstream or not conserved across species. A putative chloride ion channel gene (ClC-6) was located in the opposite orientation, at 3.5kb upstream of the original ATG codon, suggesting an overlap with the MTHFR gene and potential co-localization of regulatory elements. A CpG island was identified in the region of a 5' exon (43S) suggesting that a transcription start site and a promoter might be nearby. This work is relevant in understanding the regulation of this important enzyme in folate metabolism.

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Methylenetetrahydrofolate reductase (MTHFR) catalyses the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a carbon donor for the remethylation of homocysteine to methionine. A common mutation in MTHFR, 677C → T, which converts an alanine (A) to a valine (V) residue, is associated with increased plasma homocysteine and, consequently, is a risk factor for several vasculopathies. Recently, a second common mutation in MTHFR was reported (1298A → C) which converts a glutamate (E) to an alanine (A) residue in the C-terminal regulatory region. Thus study was designed to assess the in vitro and in vivo impact of this polymorphism. Site-directed mutagenesis of the human MTHFR cDNA was performed to create all four combinations of the two common MTHFR polymorphisms. Enzyme activity was assessed in a bacterial expression system. / Since the 1298A → C polymorphism is due to a mutation in the regulatory domain, a parallel study was performed involving the complete deletion of this region. / For in vivo assessment of the 1298A → C variant, we examined this mutation in three clinical populations. / Betaine-homocysteine methyltransferase (BHMT) is a second remethylation enzyme that converts betaine and homocysteine to dimethylglycine and methionine, respectively. This work represents the first report of sequence variants in the newly-cloned BHMT gene. Using SSCP analysis, three common base changes were identified in a panel of healthy controls. (Abstract shortened by UMI.)

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Reprints of the author's previously published articles included as an appendix. Bibliography: leaves 165-188. "This thesis describes a series of experiments that aimed to investigate the effects of folic acid deficiency and defects in folate metabolism on chromosome damage rates in human lymphocytes. The accumulation of chromosome damage over time is an important issue because it is thought to contribute to the mechanism of ageing and the aetiology of diseases of age such as cancer and Alzheimer's disease."

Reprints of the author's previously published articles included as an appendix. / Bibliography: leaves 165-188. / xiv, 189 leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / "This thesis describes a series of experiments that aimed to investigate the effects of folic acid deficiency and defects in folate metabolism on chromosome damage rates in human lymphocytes. The accumulation of chromosome damage over time is an important issue because it is thought to contribute to the mechanism of ageing and the aetiology of diseases of age such as cancer and Alzheimer's disease." / Thesis (Ph.D.)--University of Adelaide, Dept. of Physiology, 2002?