Alternative splicing of a precursor-mRNA encoded by the Chlorella sorokiniana NADP-specific glutamate dehydrogenase gene yields mRNAs for precursor proteins of isozyme subunits with different ammonium affinities.
Chlorella sorokiniana has seven ammonium-inducible, chloroplastic NADP-specific glutamate dehydrogenase (NADP-GDH) isozymes composed of varying ratios of alpha- and beta-subunits. Southern blot and allele-specific PCR analyses indicate that the C. sorokiniana genome possesses a single 7178 bp nuclear NADP-GDH gene. cDNA cloning and sequencing, 5'-RACE-PCR analysis, and RNase protection analysis identified two NADP-GDH mRNAs that are identical with the exception of a 42 nt sequence located within the 5'-coding region of the longer mRNA. The 42 nt sequence, termed an auxon because it serves as an exon or intron, appears to undergo alternative splicing from the precursor mRNA by a process that is regulated by both nutritional and environmental signals. Depending upon whether the auxon is included or excluded in a mature mRNA, the gene can be considered to consist of 22 or 23 exons, respectively. The 2074 and 2116 nt mRNAs encode precursor proteins of 56,350 and 57,850 Da, respectively. The N-termini of the purified mature alpha- and beta-subunits were sequenced, identifying full-length subunits of 53,501 and 52,342 Da, respectively. The sequences of the subunits are identical except for an 11 amino acid extension at the N-terminus of the alpha-subunit. The alpha-subunit has an additional alpha-helical domain at its N-terminus compared with the beta-subunit. By correlating the abundances of the two mRNAs with the levels (and relative turnover rates) of the alpha- and beta-subunit antigens during induction in Chlorella, the larger mRNA is proposed to encode the larger subunit.
Miller
P W
PW
Department of Microbiology and Cell Science, University of Florida, Gainesville 32611, USA.
Dunn
W I
WI
Schmidt
R R
RR
D017398:Alternative Splicing / Q000502:physiology*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002708:Chlorella / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D018086:Glutamate Dehydrogenase (NADP+) / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D008969:Molecular Sequence Data; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D017433:Protein Structure, Secondary; D000644:Quaternary Ammonium Compounds / Q000378:metabolism* / Q000494:pharmacology; D012322:RNA Precursors / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015183:Restriction Mapping; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D007527:Isoenzymes; D011498:Protein Precursors; D000644:Quaternary Ammonium Compounds; D012322:RNA Precursors; D012333:RNA, Messenger; D004247:DNA; D018086:Glutamate Dehydrogenase (NADP+)