Plastid ORF170 --Jaakko Kangasjarvi, Andrew McCullough and Burle Gengenbach In previous studies (MNL 63:105, 1989), we have found that plastid transcript accumulation patterns in maize endosperm total RNA differed significantly from patterns in leaf total RNA. Of specific interest is a ca. 2400 nucleotide transcript identified on RNA gel blots by hybridization to BamHI restriction fragment 14 (Rodermel and Bogorad, MNL 63:155, 1989) that we found to be more abundant in endosperm than in leaf total RNA. Other transcripts homologous to BamHI 14 were less abundant in endosperm. The 2400 nucleotide transcript accumulated progressively during endosperm development and became significantly more abundant between 8 and 12 DAP similar to the pattern for accumulation of spliced and unspliced transcripts for the rps12 gene (MNL 63:105, 1989).

No genes are known to be located on the BamHI 14 region in maize, but tobacco, Marchantia, rice and cauliflower have highly homologous open reading frames (167, 168, 170, 169 amino acids, respectively) in the corresponding region. Tobacco, rice and cauliflower ORFs have three exons (120 to 250 bp) separated by two type II introns (both about 750 bp). In Marchantia one of the introns is deleted. In tobacco and rice, the DNA homology extends into the introns and both introns contain an in-frame stop codon for exons I and II. Both exons I and II also begin with ATG. No function is known or has been proposed for the hypothetical protein.

To determine whether maize BamHI 14 fragment also has an ORF with similar structure, two oligonucleotide primers were designed based on the rice sequences from regions that were most homologous with tobacco and Marchantia DNA sequences from the 5' (exon I) and 3' (exon III) regions of the coding sequence. The primers directed DNA synthesis towards each other on opposite strands. RNA was isolated from maize and rice leaves and roots and 16 DAP maize endosperm. cDNA was synthesized using the 3' oligonucleotide as the primer. Then the cDNA population was amplified by polymerase chain reaction (PCR) procedures using both primers. The products were separated on an agarose gel. Three products of about 1.9, 1.2 and 0.47 kb were obtained, which corresponded in size to that expected for the unspliced transcript, to transcripts with one of the two introns spliced, and to the mature message, respectively. The 0.47 kb product from the mature transcript was detected by PCR in all tissues tested and was the only transcript detected in leaves. The unspliced 1.9 kb product was the most abundant PCR product in endosperm. In the control experiment where leaf DNA was used for the PCR template, only the 1.9 kb fragment was amplified as expected for the intact unspliced sequence. The BamHI 14 fragment hybridized to all three amplified products indicating their homology with maize plastid DNA. The amplified 0.47 kb product hybridized to a 2400 nucleotide transcript on gel blots of endosperm RNA.

Sequence data banks were searched for homology to the ORF170 sequence. No homology at the amino acid sequence level was identified when each exon from rice was used separately. When shorter 100% conserved amino acid sequences between tobacco, rice and Marchantia were used, however, we found homology to several proteins. The most common feature of these proteins was a function relating to some type of recognition or interaction with DNA. This possibility is being tested with the cloned sequence.


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