Rider University

Identification of two cDNAs encoding methylenetetrahydrofolate reductase --Khan, F, Kite, M, Benner, M Synthesis of methionine in higher plants involves the net interaction of three processes: 1) formation of an aspartate-derived, four-carbon chain, 2) transfer of a sulfur atom derived from cysteine, and 3) addition of a methyl group donated by 5-methyltetrahydrofolate. Although the enzymes required for these three processes have been identified, the genes encoding them are largely uncharacterized in higher plants. The methyl donor 5-methyltetrahydrofolate is synthesized from 5,10-methylenetetrahydrofolate by the enzyme methylenetetrahydrofolate reductase (MTHFR); this enzyme is critical for the maintenance of an adequate methionine pool in both eukaryotes and prokaryotes. The mammalian genes have been shown to encode two domains - a catalytic domain which binds the substrate and a regulatory domain which contains an allosteric site for S-adenosylmethionine binding (Goyette et al., Nature Genetics 7:195-200). We wish to investigate the role of the regulatory domain in higher plants.

BLAST searches were performed to identify plant ESTs that showed significant homology with previously identified MTHFR sequences. Alignment of the ESTs and comparison with the human cDNA facilitated the design of a degenerate oligonucleotide primer that anneals near the 5 end of the maize MTHFR cDNA and includes the start codon. A primer sequence for the 3' end of the cDNA was designed from a maize EST which contains the putative stop codon. RNA was extracted from maize leaf tissue obtained from two-week old seedlings of W64A using the Qiagen RNeasy protocol. cDNA was prepared with the Qiagen Omniscript RT system. MTHFR cDNA was amplified via 35 cycles of 95 C (30 sec), 50 C (30 sec), and 72 C (2 min). The purified PCR products were ligated into the Stratagene PCR-Script Amp cloning vector. Ligation products were used to transform E. coli strain DH5a; clones were independently sequenced at Rider University and Princeton University.

As predicted, utilization of the above primers in PCR reactions containing maize cDNA resulted in the amplification of a single fragment of approximately 1.8 kb. Sequence analyses indicate that we have isolated two members of the MTHFR gene family in maize. One of our clones is identical to a recently submitted sequence (Roje et al., J. Biol. Chem. 274: 36089-36096), while the other is unreported. Presence of more than one locus has been suggested by Roje et al.; in addition, the RFLP marker csu134 has been show to be homologous to MTHFR sequences and hybridizes to two thf loci, one mapped to chromosome 1 (csu134a) and one mapped to chromosome 5 (csu134b) (Chao et al., Theor Appl Genet 88:717-721). We hypothesize that one of our clones represents the chromosome 1 locus, while the other represents the chromosome 5 locus. To date, we have ascertained that the unreported cDNA sequence differs with respect to at least three nucleotides. Two of these differences presumably result in divergence at the amino acid level as well (T59A; L132F). In addition, one of the polymorphisms alters a restriction site, giving us a gene-specific difference that will facilitate the confirmation of map location.

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