Chlorella sorokiniana has seven ammonium-inducible, chloroplastic NADP-specific glutamate dehydrogenase (NADP-GDH) isozymes composed of varying ratios of α- and β-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 56350 and 57850 Da, respectively. The N-termini of the purified mature α- and β-subunits were sequenced, identifying full-length subunits of 53501 and 52342 Da, respectively. The sequences of the subunits are identical except for an 11 amino acid extension at the N-terminus of the α-subunit. The α-subunit has an additional α-helical domain at its N-terminus compared with the β-subunit. By correlating the abundances of the two mRNAs with the levels (and relative turnover rates) of the α- and β-subunit antigens during induction in Chlorella, the larger mRNA is proposed to encode the larger subunit.
Plant Molecular Biology – Springer Journals
Published: Oct 6, 2004
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