Expression of zein genes and possible intron structure(s)

The expression of zein genomic clones has been studied in our lab in in vitro transcription systems (Hela cell and Xenopus oocyte germinal vesicle extract systems: P. Langridge and G. Feix, Cell 34:1015-1022, 1983) and in vivo in yeast (P. Langridge et al., EMBOJ. 3:2467-2471,1984) and Acetabularia. In particular, the Acetabularia system has proved to be extremely valuable as zein genes are expressed to the protein level. Essentially, supercoiled plasmid DNA of zein genomic clones was injected into isolated Acetabularia nuclei. These nuclei were reimplanted into anucleated Acetabularia cells. After incubation, cytoplasmic smears were reacted with zein antibodies which were then reacted with an anti-IgG-fluorescein conjugate. Positive results, as detected by immunofluorescence, were observed for a number of genomic clones. Acetabularia is, therefore, a very valuable in vivo test system for the functionality of isolated zein genes.

In one case an isolated gene which gave a strong positive result in Acetabularia does not have the full reading frame. According to DNA sequence analysis, an insertion of an adenosine residue at + 140 has occurred resulting in a termination codon after 48 amino acids. Such a clone would normally be looked upon as a "pseudogene"; however, due to the result in Acetabularia, this conclusion must be re-examined. Either a truncated protein which is still recognised by zein antibodies is produced, or some zein genes contain intron structures to overcome premature termination of translation. Until now the zein gene family has been thought to contain no intron structures. S1-mapping in the region of the single base insertion has shown S1 signals which may represent the borders of an intron. If this is the case, the intron(s) which may occur in some zein genes are postulated to be small (< 50 bases) as they have not been detected by electron microscopic R-loop analysis.

J.W.S. Brown, P. Langridge and G. Feix

Please Note: Notes submitted to the Maize Genetics Cooperation Newsletter may be cited only with consent of the authors.

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