ABSTRACT
The isolation and genomic sequence of one of possibly four glyceraldehyde-3-phosphate dehydrogenase genes in the nematode, Caenorhabditis elegans is presented. The complete nucleotide sequence of the coding as well as the noncoding flanking regions of this gene has been determined. The deduced amino-acid sequence agrees with the sequence of typical glyceraldehyde-3-phosphate dehydrogenase enzymes and its molecular weight of 36,235 agrees with its size determined previously (Yarbrough, P. and Hecht, R. (1984) J. Biol. Chem. 259, 14711-14720). That this isolated gene encodes a nematode glyceraldehyde-3-phosphate dehydrogenase is additionally confirmed by demonstrating its immunoreactivity to an anti-nematode glyceraldehyde-3-phosphate dehydrogenase antibody after its expression as a fusion protein with dihydrofolate reductase. Codon utilization follows a pattern typical of other expressed nematode genes. The gene is split by two introns that are highly conserved in comparison to other introns observed in C. elegans. The placement of one of these introns is conserved with respect to the chicken glyceraldehyde-3-phosphate dehydrogenase gene. Within the 5' flanking sequence homology to actin and the homology 2 block of the major myosin gene (unc-54) is noted. It is of interest that the 3' flanking region contains a CAAAT box, followed by a TATAAT box, before an open reading frame of a closely linked gene that also contains a small AT-rich intron with the nematode consensus splice junction.
Subject(s)
Caenorhabditis/genetics , Glyceraldehyde-3-Phosphate Dehydrogenases/genetics , Amino Acid Sequence , Animals , Base Sequence , Binding Sites , Biological Evolution , Cloning, Molecular , DNA Restriction Enzymes , GenesABSTRACT
Two glyceraldehyde-3-phosphate dehydrogenases have been separated and purified from the nematode Caenorhabditis elegans. As defined by starch gel electrophoresis, the faster-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-2, increases its activity during postembryonic development. In contrast, the slower-migrating isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is enriched in isolated embryos. Both isoenzymes were initially purified by ammonium sulfate fractionation, gel filtration, and NAD+-agarose affinity chromatography. The separation of both isoenzymes as well as their purification to homogeneity was obtained by preparative chromatofocusing. The subunit molecular weight of each isoenzyme is 38,500 +/- 500. A tetrameric native molecular weight of 157,000 +/- 2000 was determined for glyceraldehyde-3-phosphate dehydrogenase-2. Monospecific rabbit polyclonal antibodies were initially raised against the major isoenzyme and subsequently used to characterize both isoenzymes. Staphylococcus aureas V8 protease digests of each isoenzyme were separated electrophoretically and stained immunochemically, providing evidence that the two isoenzymes differed in their amino acid sequences. Developmental immunocytochemical studies suggest that the embryonic-enriched isoenzyme, glyceraldehyde-3-phosphate dehydrogenase-1, is present in all cells. The second isoenzyme, exhibiting the major activity during postembryonic larval development, may define a body-wall-muscle specific activity which is located within the actin-containing I and A zones of the nematode's sarcomeres.
