Transposon tagging of the indeterminate gene
--Joseph Colasanti and Venkatesan Sundaresan

The indeterminate (id1) mutation of maize causes a prolonged vegetative phase of development that results in an increase in the number of leaves and a late flowering phenotype. In a previous issue of the Maize News Letter (MNL 66:30) we reported the isolation of a new indeterminate mutation, id*, and suggested that this mutation was the result of a Ds2 transposon insertion. Since then we have shown that id* is allelic to two known id1 mutations, id1-R (from the Maize Coop) and id1-Compeigne (courtesy of Ben Burr, Brookhaven); we designate this new allele as id1-CSH. The id1-CSH allele was crossed into several different backgrounds and molecular analysis was performed on these plants to determine if a Ds2 element segregated with the id phenotype.

A clone containing a 100 bp DNA fragment specific to Ds2 (gift of Sarah Hake, U.C. Berkeley) was used to probe Southern blots. In over 120 progeny analysed, a 4.2 kb SacI fragment was found that was always present in plants that carried the id1-CSH allele, and was absent in plants that did not carry it. This fragment was cloned into a pUC-based plasmid, pLITMUS29, and restriction mapped. The Ds2 element was inserted 165 bp from one end of the 4.2 kb fragment (the "right flank") and 2.9 kb from the other end ("left flank"). When part of the left flank DNA was used to re-probe the original Southern blots, a single 4.2 kb SacI fragment was found in id1-CSH homozygous plants and a 2.9 kb SacI fragment was found in plants that did not have the id1-CSH allele; heterozygous plants had both bands. Interestingly, when left or right flank DNA was used to probe SacI cut DNA from id-R plants, no band was visible. This suggests that the id1-R allele might be a deletion of part or all of the Id1 gene. Further, the id1-Compeigne allele, when probed with the flanking DNA, exhibited a pattern which suggests that an insertion of approximately 3 kb exists in the left flank region. Taken together, these results strongly suggest that the id1-CSH mutation is the result of a Ds2 insertion.

Sequence analysis of the regions immediately flanking the Ds2 element revealed that the transposon is inserted into a putative open reading frame (ORF) of at least 84 amino acids. Preliminary RNA blot analysis using a DNA fragment containing part of the ORF region as a probe showed that it hybridized to a relatively abundant transcript of approximately 2.0 to 2.2 kb in polyA+ RNA from shoot apex tissue, and, to a much lesser extent, in mature leaf tissue. Very little hybridization was detected in seedling RNA, and no transcript was detected in RNA from roots. Comparison of the ORF sequence to known sequences in the database showed that it has similarities to "zinc-finger" DNA binding proteins from several species, including humans, Drosophila and Xenopus. This is further suggestive evidence that, if the ORF is a part of the Id coding sequence, the Id protein might function as a regulator of the vegetative-to-reproductive transition. 


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