Isolation and characterization of the messenger RNA and the gene coding for a proline-rich zein from corn endosperm

Gamma-zein, a proline-rich protein from corn endosperm, was investigated at the molecular level. Immunological and electrophoretic data indicated that gamma-zein was deposited into protein bodies in corn endosperm. Both isolated polysomes and poly(A)⁺ mRNA were found to direct in vitro synthesis of gamma-zein in a wheat germ system. In vitro synthesized gamma-zein was immunoprecipitated from the total in vitro translation products. A cDNA expression library was constructed by reverse transcription of total poly(A)⁺ mRNA using pUC8 plasmid as vector and E. coli strain DH1 as host. The library was screened for the expression of gamma-zein and alpha-zein by specific antibodies. The library was also screened with ³²P-labeled gamma-zein and alpha-zein cDNA probes. The results indicated that gamma-zein and its fragments were readily expressed in E. coli while alpha-zein was not.

Seven independently selected clones, six of which were selected by antibody and one by a cDNA probe, were sequenced. A comparison of sequence information from seven clones revealed that their overlapping regions were identical. This suggests that gamma-zein is encoded by a single U gene. This finding is in conflict with what was expected on the basis of extensive charge heterogeneity of gamma-zein in isoelectric focusing. Individual bands cut from an IEF gel were rerun and shown to give several bands suggesting that the charge heterogeneity of gamma-zein may be an artifact. Sequence information of gamma-zein indicated that the gene encodes a mature protein whose primary structure includes 204 amino acids and has a molecular weight of 21,824 daltons. There are eight essentially identical tandem repeats of the hexapeptide Pro-Pro-Pro-Val-His-Leu and two of the octapeptide Gln-Pro-His-Pro-Cys-Pro-Cys-Gln in the N-terminal one-half of the polypeptide. The codon specifying the third proline in the hexapeptide repeating unit is identical, CCG, in all eight repeats. It is likely that these highly conserved tandem repeats are of critical importance to the function of gamma-zein which is presently unknown. Alternatively, it is conceivable that selective pressures responsible for conserving these tandem repeats may be operating at the nucleic acid level.