The 250bp cDNA clone (PCM4), previously isolated from a cDNA library of polyA-RNA of maize endosperm polysomes, shown to be localized by in situ hybridization on the long arm of chromosome 5 and represented in endosperm polyA-RNA by 4 RNA species ranging from 1.5 to 4.5Kb in length, has been further characterized. In cDNA libraries from various maize tissues, M4 related clones represent about 1-3% of the total recombinant phages and their abundance in the endosperm cDNA library is comparable to that of zein sequences.

The M4 sequence does not show significant homology either to maize storage protein genes or to other published maize sequences. Southern blot analysis showed that the genomic region containing the M4 sequence is highly conserved into 2 unrelated maize lines, W64A and A69Y. The resulting hybridization pattern of the genomic DNA digested with several restriction enzymes (EcoRI, XbaI, BamHI, HindIII, EcoRV) is exactly the same in these two maize lines. Other organisms have been analyzed for the presence of M4 related sequences; in particular, some related plants (teosinte, oat, barley, wheat), some dicots (petunia, bean, tobacco), and some unrelated organisms like yeast, rat and man. Their DNAs have been digested with EcoRI and HindIII and hybridized with the M4 fragment. The EcoRI digestion gives rise to a single hybridization band (about 8Kb in the monocots and about 3.5Kb in the dicots) while HindIII digestion results in 2 bands (of similar molecular weight in monocots, different weight in dicots) as a HindIII site is contained in the probe. No hybridization signals were detectable in yeast, man and rat. Copy number evaluation suggests the presence of 1-2 copies per maize haploid genome.

Northern blot analysis of either total or polyA-RNA from different maize tissues reveals the existence in all the RNA investigated of the 4 M4 transcripts previously shown in endosperm RNA. The relative abundance of each of the 4 RNA molecules is different in the various tissues in which one or two of the transcripts are barely detectable. A similar transcript pattern is also present in petunia and in bean tissues. The transcripts are identical in size in the two dicots but different from the maize M4-related RNAs. Studies are in progress to elucidate the tissue-specific regulation of the 4 transcripts, and genomic clones containing the M4 sequence are under characterization.

F. Quattrocchio, F Sparvoli and A. Viotti

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