Analysis of a partial Adh1-3F1124 germinal revertant --Brandon DeFrancisci, Barbara Kloeckener-Gruissem and Michael Freeling In order to learn about the activity, excision and transposition of the Mu3 transposable element and to gain information about the promoter region of Adh1, revertants of the mutant Adh1-3F1124 were isolated. These revertants were isolated from Adh1-3F1124/Adh1-2F11 heterozygous seeds. Adh1-3F1124 is a conditional anaerobic lethal mutant caused by the insertion of a 1.85kbp Mu3 transposon resulting in the duplication of the TATA box (C-H. Chen et al., Genetics 116:469-477). It has 6% of wild type ADH1 activity in primary roots and scutellum but normal activity in pollen. Adh1-2F11 has no ADH1 activity and is caused by the insertion of Ds (Doring et al., MGG 193:199-204). Over one hundred thousand seeds were screened under partially anaerobic conditions. Six Adh1-3F1124 revertants were recovered.

One of these revertants, Adh1-3F1124-r53, has 50% of wild type ADH1 activity in the scutellum and normal activity in the pollen. Using Adh1 and Mu3 specific probes, Southern analysis indicates the expected restriction sites for the Adh1-3F progenitor allele and that Mu3 has excised. Approximately 100bps surrounding the excision site were sequenced after amplification of this DNA fragment using the polymerase chain reaction (PCR) according to Perkin Elmer Cetus. The sequence was found to be identical to the progenitor allele, Adh1-3F. To see whether the phenotype of r53 is linked to Adh1-3F1124 mutant allele, linkage analysis was done by crossing Adh1-3F1124-r53/Adh1-1S heterozygotes to Adh1-3F1124 homozygotes. ADH1 activity in the scutellum segregated 1:1 for Adh1-1S/Adh1-3F1124 and Adh1-3F1124-53/Adh1-3F1124 indicating linkage between the revertant and mutant allele. In order to determine the cause of r53's phenotype, the allele will be further examined by thermostability studies of the protein and by obtaining and analyzing a large genomic clone.


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