Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott).
Polyclonal and monoclonal antibodies that recognize the 35-, 36-, and 37-kDa alternative oxidase proteins of Sauromatum guttatum (Schott) were used to isolate a cDNA clone, pAOSG81, from an S. guttatum cDNA expression library. A fusion protein with an apparent molecular mass of 48 kDa was expressed from a pUC119 derivative of pAOSG81 (pAOSG81-119) in Escherichia coli cells and was recognized by the monoclonal antibodies. When the in vitro translated and immunoprecipitated products made from mRNA hybrid-selected by pAOSG81 were analyzed, a single band corresponding to a protein with an apparent molecular mass of 42 kDa was observed. DNA sequence characterization showed that pAOSG81 contains the entire coding region of a protein with a calculated molecular mass of 38.9 kDa, a putative 63-amino acid transit peptide, and a 9-amino acid match to the authentic N-terminal sequence of the 36-kDa alternative oxidase protein. Analyses of the deduced amino acid sequence indicate: (i) that the transit peptide is predicted to form amphiphilic helices, and (ii) that three regions of the processed protein are likely to form transmembrane alpha-helices. We conclude from these data that pAOSG81 represents a nuclear gene, aox1, encoding a precursor protein of one or more of the alternative oxidase proteins of S. guttatum.
Rhoads
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DM
Michigan State University, Department of Energy Plant Research Laboratory, East Lansing 48824.
McIntosh
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D024101:Mitochondrial Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D010940:Plant Proteins; D010944:Plants / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D014176:Protein Biosynthesis; D011487:Protein Conformation; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D000099188:Alternative Oxidase
D004247:DNA; D024101:Mitochondrial Proteins; D010088:Oxidoreductases; D010940:Plant Proteins; D011061:Poly A; D012313:RNA; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D000099188:Alternative Oxidase