Preliminary characterization of a derivative allele of an unstable regulatory mutant at Adh1

A Robertson's Mutator-induced mutant at Adh1, Adh1-S3034, has been characterized as low in expression of enzyme activity, protein (CRM), and mRNA to approximately 40% of the wild type level. It is also genetically unstable: pollen stained for ADH activity shows grains with greater and lower levels of ADH. Two derivative alleles , S3034a and S3034b, having 0% and 13% levels of enzyme activity and mRNA relative to wild type Adh1-S, have been described previously (Freeling, Cheng and Alleman, 1982, Devel. Genet., in press; Strommer, Hake, Bennetzen, Taylor and Freeling, 1982, Nature 300:542). A 1.35 kb insert (Mu1) in S3034 is unchanged in the derivatives at the level of resolution possible by restriction site mapping studies.

We have selected a derivative allele of S3034 which is altered in the protein produced by the Adh1 gene. This derivative, Adh1-S3034x1, produces a product which forms an active dimer with the electrophoretic variant allozyme subunit ADH1-F. The allozyme profile of Adh1-S3034x1/Adh1-F heterozygotes show that the S3034x1.F dimers migrate to the same position as S.F dimers. The allozyme ratio as well as CRM levels in the mutant homozygotes implies that 15-20% product is formed. S3034x1 homozygotes, however, have no detectable ADH enzyme activity.

Unpublished data of Hake, Taylor, Strommer and Bennetzen suggested that the inserts of Adh1-S3034 and the derivatives a and b are in the first intervening sequence of the gene; sequence information verified this (intron sequence communicated by W. J. Peacock; point of insertion is from unpublished sequence data from J. Bennetzen and J. Strommer). Since none of these alleles have been shown to alter the protein product, it was thought that the derivative S3034x1 could test for transposition of the Mu1 element. Gross transposition is not the case because of the following data: restriction site mapping shows that S3034x1 maps to the same location as S3034, a and b and has a BstEII site in the same place within the insert. There are no additional inserts elsewhere in the gene. The level of resolution of restriction site mapping using the size of DNA fragments generated in these experiments is not great (approximately 50 b.p.). Since the intron/exon junction nearest the Mu1 insertion is close, changes that could alter coding information but which could not be detected by genomic southerns is possible. Although small scale rearrangements around the Mu1 insertion site have not been ruled out by these tests, simple transposition or excision of the element has. One of the many other derivatives that remain untested may provide the evidence for transposition.

It is easy to concoct hypotheses that could explain how insertions within an intron could lead to different mRNA levels. How Mu1 has come to affect coding sequence is more difficult to understand.

Mary Alleman and Michael Freeling

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