Rat C1-tetrahydrofolate synthase. cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast.
We have isolated and characterized cDNA clones encoding rat cytoplasmic C1-tetrahydrofolate (H4folate) synthase. In eukaryotes, this enzyme is trifunctional and contains the activities of 10-formyl-H4folate synthetase, 5,10-methenyl-H4folate cyclohydrolase, and 5,10-methylene-H4folate dehydrogenase. The deduced sequence of the 935-amino acid open reading frame contained exact matches to NH2-terminal (15 residues) and internal (residues 436-450) peptide sequences obtained from the purified enzyme. The amino acid sequence derived from the rat cDNA shows extensive homology to analogous proteins from bacterial, yeast, and mammalian sources. We have used the cDNA to determine the steady-state levels of the mRNA in various rat tissues and have found that gene expression is regulated in a tissue-specific manner. Transcript levels are highest in kidney and liver with liver transcripts reduced about 30% relative to those found in kidney. Brain, heart, testis, lung and skeletal muscle display even lower transcript levels; reductions range from 70 to 80% of transcript levels found in kidney. Comparison to the levels of enzyme in these tissues allows us to conclude that pretranslational events predominate in the tissue-specific expression. The rat enzyme has been functionally expressed in Saccharomyces cerevisiae as evidenced by its capacity to complement a chromosomal deletion of ADE3, the yeast gene encoding cytoplasmic C1-H4folate synthase.
Clayton Foundation Biochemical Institute, Department of Chemistry, University of Texas, Austin 78712.
A E
AE
Thigpen
M G
MG
West
D R
DR
Appling
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005574:Formate-Tetrahydrofolate Ligase / Q000235:genetics*; D015870:Gene Expression; D007668:Kidney / Q000201:enzymology; D008025:Ligases / Q000235:genetics*; D008099:Liver / Q000201:enzymology; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010088:Oxidoreductases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid; D014018:Tissue Distribution; D014158:Transcription, Genetic
D009097:Multienzyme Complexes; D012333:RNA, Messenger; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D004247:DNA; D010088:Oxidoreductases; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D008025:Ligases; D005574:Formate-Tetrahydrofolate Ligase