A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) inChlorella sorokiniana

Abstract

Chlorella sorokiniana possesses ammonium-inducible, chloroplastic, NADP-specific glutamate dehydrogenase (NADP-GDH) homo- or heterohexamers composed of alpha- and/or beta-subunits which were previously shown to derive from precursor protein(s) of identical size. From the present studies, data are consistent with these two subunits being encoded by a single nuclear gene. The NADP-GDH gene is greater than 7 kb in length due to the presence of at least 21 introns, an unusually large number for a eukaryotic microorganism. The exons, identified by comparison with sequences of NADP-GDH cDNA clones, include a region which is highly conserved among NADP-GDH genes. This region in the C. sorokiniana gene is 77% and 73% identical to the corresponding regions in the Escherichia coli and Neurospora crassa NADP-GDH genes, respectively. Seventeen independent NADP-GDH cDNA clones were analyzed by restriction mapping and partial sequencing, and no differences were detected among them. The longest cDNA was fused in frame with lacZ in a Bluescript vector and was expressed in E. coli as NADP-GDH antigen. During a 240 min induction period, under conditions in which both types of subunits were synthesized, only a single (2.2 kb) NADP-GDH mRNA band was detected on northern blots using cDNA probes from the highly conserved and 3'-untranslated regions. Collectively, these results are consistent with a single mRNA encoding a precursor-protein which is differentially processed to yield either an alpha- or beta-subunit.