A nuclear gene with many introns encoding ammonium-inducible chloroplastic NADP-specific glutamate dehydrogenase(s) in Chlorella sorokiniana.
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.
Cock
J M
JM
Department of Microbiology and Cell Science, University of Florida 32611.
Kim
K D
KD
Miller
P W
PW
Hutson
R G
RG
Schmidt
R R
RR
D000595:Amino Acid Sequence; D000641:Ammonia / Q000494:pharmacology*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000502:physiology*; D002708:Chlorella / Q000187:drug effects / Q000201:enzymology / Q000235:genetics*; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004790:Enzyme Induction; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D005796:Genes; D005969:Glutamate Dehydrogenase / Q000096:biosynthesis / Q000235:genetics*; D007438:Introns; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009492:Neurospora crassa / Q000201:enzymology / Q000235:genetics; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid
D003062:Codon; D046911:Macromolecular Substances; D012333:RNA, Messenger; D012313:RNA; D000641:Ammonia; D004247:DNA; D005969:Glutamate Dehydrogenase