Genetic and biochemical evidence indicates that the zein proteins are coded by a large number of genes, although estimates of the exact number still vary greatly. In order to analyze the organization and expression of these genes it is necessary to isolate genomic clones that will contain only one or a few zein genes. We have previously reported the isolation and characterization of one such zein gene (Mol. Gen. Genet. 182:440-444, 1981) but we now have several new and larger clones.
Zein coding sequences were identified from a maize DNA bank of Eco RI restricted seedling DNA of variety A619 cloned into lambda gt WES. cDNA clones specific for either the 19,000 or 21,000 dalton protein classes were used as probes for selecting the genomic clones. This method has yielded so far about 50 zein genomic clones with maize inserts varying in size from less than 2 thousand to nearly 14 thousand base pairs. After subcloning into pBR 328 we have analyzed 6 of these fragments in detail: 2 for the 19,000 dalton proteins and 4 for the 21,000 dalton size class.
The following types of analyses have been employed to gather information about the zein genes.
(a) Electron microscopic observation of hybrids formed between the genomic clones and endosperm poly rA+RNA (R-loops) and the cDNA clones (D-loops): These experiments demonstrate that all clones contain a complete zein gene with no detectable intervening sequences.
(b) Mapping of restriction endonuclease recognition sites has shown that the zein genes contain few sites relative to their flanking sequences. It has also been found that all 6 cloned fragments differ from each other.
(c) Hybridization of the genomic clones with restriction endonuclease treated and electrophoretically fractionated total maize DNA (Southern analysis) has revealed patterns similar to those shown when the cDNAs are used as hybridization probes. Each of the genomic clones gives the pattern characteristic for its specific cDNA. This indicates that the flanking sequences do not contain major repetitive sequences. It has also been found that the sequences flanking the zein genes show some homology between clones but there are also some areas that do not cross-hybridize.
It will be necessary to analyze further clones in order to obtain a more general picture of zein gene structure and organization. It is significant that of 6 clones analyzed by us, all differ, to varying degrees, from one another. The identification and interpretation of the mechanisms controlling zein expression will therefore probably be complex and involve considerable further effort.
J. Pintor-Toro, P. Langridge and G. Feix
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