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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