Amplification of the methylenetetrahydrofolate reductase gene
--Michael Benner, James Johnson, Michael Weisberg, Deb Jones and Donna Cartledge
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which in turn donates a methyl group for the conversion of homocysteine to methionine. Genes encoding this enzyme have been isolated from E. coli, S. typhimurium, Saccharomyces cerevisiae and, most recently, humans. Alignment of prokaryotic and eukaryotic amino acid sequences reveals several areas of homology; we hypothesized that these regions of homology also exist in higher plants. We have designed degenerate oligonucleotide primers that result in the successful amplification of a putative maize MTHFR gene.
Degenerate primers corresponding to the +76 to +95 region and the +338 to +356 regions of the E. coli metf sequence were synthesized. The amino acids encoded by these regions correspond in position to those found in the N-terminal 40 kDa domain of the human enzyme. The predicted length of the E. coli metf gene fragment amplified by these primers is 281 bp; the predicted length of the amplified human gene sequence is 293 bp.
Utilization of the above primers in PCR reactions containing maize genomic DNA results in the amplification of a single major fragment of approximately 270 bp. When the maize amplification product is radiolabeled and used to probe genomic DNA, a single major band is evident. We are currently isolating a full-length clone for subsequent sequence and expression analyses. The availability of a maize MTHFR clone will facilitate the investigation of methionine production in higher plants.
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