Zein messenger RNAs from maize endosperm were purified by successive oligo(dT)-cellulose chromatography and sucrose gradient centrifugation. Polyacrylamide gel electrophoresis under denaturating conditions revealed the presence of two size classes of zein messenger RNAs of M , 3.5 x lo5 and 4.10 x lo5. The mRNA was shown to synthesize the major zein polypeptides, to have a base composition characteristic of a poly(A)-containing RNA and to be transcribed by reverse transcriptase into complementary DNA. The yo t l j z of the hybridization curve of cDNA hybridized to an excess of mRNA was shown to be 7 x l o p L M . s indicating that about 15 non-cross-hybridizing sequences are present in the zein mRNA preparations. The kinetics of cDNA annealing with an excess of maize DNA from 2n cells suggest a ten-times reiteration of each mRNA sequence. This result is confirmed from saturation experiments, where in cDNA excess to DNA, the number of zein genes per haploid maize genome was estimated as about 120 copies. Similar experiments carried out on DNA from normal and mutant endosperms (3n cells) indicate the absence of large amplifications or deletions of zein genes in the tissue devoted to zein synthesis.During development, maize endosperm accumulates a large amount of storage proteins [1,2]. The main protein fraction is zein, a hydrophobic protein synthesized by membrane-bound polysomes and stored in granules (protein bodies) [3 -51.The hydrophobicity of zein is due to its high level of neutral and hydrophobic amino acid residues (more than 90%) and to the low level of basic amino acids such as lysine, which also contribute to the low biological value of maize seed proteins [6-111. As shown by dodecylsulfate electrophoresis, zein consists of between three and five major polypeptides in the 22 700 -19 000 molecular weight range and some minor components of M , 16000-14000 [12,13]. A wider heterogeneity (8-15 components) can be obtained by urea/acrylamide gel electrophoresis [14,15] or by isoelectric focusing [I 21. Bidimensional analysis (isoelectric focusing/dodecylsulfate electrophoresis) shows that some of the isoelectric-focusing zein bands behave as single polypeptides, while others can be further resolved into two or three components [16]. Altogether, the data indicate that zein is fractionable into at least 20-25 polypeptides [17,18]. The isoelectric-focusing pattern of zein was shown to be genotype-specific [8, 121 and not due to post-translational modifications [5]. Moreover, genetic studies have demonstrated that the zein isoelectric-focusing components behave as direct products of structural genes [19]. In order to establish the molecular basis for such a wide heterogeneity and to obtain further data on the genetic system controlling zein synthesis, we studied by molecular hybridization the complexity of zein messenger RNAs and the number of zein genes in the maize genome.
MATERIALS AND METHODS
MaterialsMaize seeds obtained 21 days after pollination, from the inbred line W64A grown in the field, were harvest...
About half of the spontaneous petite mutants produced by wild‐type Saccharomyces cerevisiae strain B (as well as by several other strains) have the same defective mitochondrial genome. Its repeat unit is a segment, 2200 base pairs (bp) long, which derives from an excision between the origins of replication ori 2 and ori 7 of the wild‐type genome, and contains a hybrid ori 2‐ori 7 sequence. The spontaneous petites carrying this defective ori.h genome are supersuppressive, i.e., they very rapidly compete out the wild‐type genome in crosses. The main reasons for the exceptional frequency of ori.h petites are an extremely high excision frequency, due to the extended homology between the two tandemly oriented ori sequences 265 bp long and the short distance separating them. Such an excision frequency is very strongly increased in petite genomes encompassing the ori 2‐ori 7 region, because of their higher concentration in these ori sequences.
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