Molecular analysis of multiple mutator-derived alleles of the bronze locus of maize.

Very few mutations derived from Mutator maize lines have been studied at the molecular level. The variety of Mu elements that can induce mutations, the relative frequency of mutant induction by insertion of a given class of Mu elements or by a Mu-induced genomic rearrangement, a possible intragenic insertion site specificity, and the molecular nature of reversion events are all unknown in the Mutator system. To address these questions, we have isolated several partially or fully inactivated bronze alleles from Mutator maize lines and structurally characterized them by gel blot hybridization of genomic DNA. The mutations were induced in three parental Bronze alleles which differ by polymorphisms flanking the coding region. Each of the 14 inactivated bronze mutants characterized was found to contain an insert which cross-hybridized with the transposable element Mu1. Detailed maps of 11 of these alleles revealed a 1.4-kb insert with restriction sites characteristic of Mu1. These Mu1 insertions were found dispersed throughout both of the Bronze exons and in either orientation relative to Bronze transcription. Stable and somatically unstable (mutable) mutant alleles differed with respect to the covalent modification of restriction sites within the inserted Mu1 element. Several germinal revertants of one mutable bronze allele, bzMum4, were isolated. These all were associated with excision of the Mu1 element from the affected locus.

The effects of defective Suppressor-mutator insertions on the expression of a Bronze-1 allele in maize

Molecular and biochemical analyses of the maize transposable element mutant bronze-m13, which resulted from the insertion of a defective Suppressor-mutator element in an exon of a Bronze-1 allele, and of changes of state derived from bronze-m13 by internal deletions within the element have revealed how these mutant alleles condition a nonmutant phenotype in the absence of a trans-active Suppressor-mutator. The transposable element insertions are all in the same position, 38 base pairs 3′ to the single intron present in the bz locus. The insertions are transcribed with the gene, and the pre-mRNAs of bronze-m13 and CS1, CS5, CS6, and CS12 are then spliced using the intron donor splice site and either one of two acceptor splice sites in the defective Suppressor-mutator element. Only one of these two messages is translated to yield a functional enzyme. The pre-mRNA of CS9 is spliced only in the reaction that gives a translatable message since the pre-mRNA lacks the alternate acceptor splice site. The splicing reactions are detailed and related to the very different amounts of enzymatic activity produced by these alleles. The presence of an antisense message in CS12 plants to the defective Suppressor-mutator sequence transcribed with the bronze locus is also discussed.Key words: maize transposable element, Suppressor-mutator, splicing.

Sequence of three bronze alleles of maize and correlation with the genetic fine structure.

The genomic sequences of three bronze alleles from Zea mays, Bz-McC, Bz-W22 and bz-R, are presented together with their flanking sequences. The bronze locus encodes UDPglucose flavonoid glucosyl-transferase (UFGT), an anthocyanin biosynthetic enzyme. The wild-type alleles Bz-McC and Bz-W22 condition purple phenotypes in the seed and plant, while bz-R conditions a bronze color. A full length cDNA corresponding to the Bz-McC allele was cloned and sequenced. Primer extension and RNase protection experiments were used to verify the 5' end of the bronze transcript. The Bz-McC allele has a 1416-bp coding region, a 100-bp intron and an approximately 83-bp 5' leader. Upstream of the message initiation site the sequences CTAACT and AATAAA occupy the positions where the eukaryotic consensus CCAAT and TATA boxes are normally found. The alleles Bz-McC and bz-R each have different large insertions with characteristics of transposable elements in their 5' flanking regions. The bz-R allele is distinguished by a 340-bp deletion starting within the intron and including 285 bp of the second exon. The Bz-McC and Bz-W22 isoalleles are known to differ in two genetically defined locations. The uts and uqv sites from the Bz-McC allele condition, respectively, lowered thermostability for the UFGT enzyme and increased amount of UFGT activity when compared with the corresponding sites in the Bz-W22 allele. The uts site maps to a region of the gene encoding two adjacent amino acid differences, either or both of which might alter the thermostability of the UFGT enzyme. The difference in UFGT levels conditioned by the uqv site is shown here to be correlated with variation in the bronze mRNA level. A likely cause of this decreased bronze mRNA level in Bz-W22 is a 6-bp duplication near the sequence CTAACT located 74 bp upstream of the bronze message initiation site. This region is therefore tentatively identified as the uqv site.