The bronze (bz) locus of maize has been cloned by an indirect procedure utilizing the cloned transposable controlling element Activator (Ac). Restriction endonuclease fragments of maize DNA were cloned in bacteriophage X and recombinant phage with homology to the center of the Ac element were isolated. The cloned fragments were analyzed to determine which contained sequences that were structurally identical to a previously isolated Ac element. Two such fragments were identified. Sequences flanking the Ac element were subcloned and used to probe genomic DNA from plants with well-defined mutations at the bz locus. By this means, it was established that one of the genomic clones contained a bz locus sequence. The subcloned probe fragment was then used to clone a nonmutant Bz allele of the locus. The method described here should prove useful in cloning other loci with Ac insertion mutations.The enzyme UDPglucose-flavonol glucosyltransferase catalyzes the 3-O-glucosylation of flavonols and anthocyanidins (1, 2). Studies on strains with recessive mutations at the bronze (bz) locus have provided evidence that the locus encodes the UDPglucose-flavonol glucosyltransferase enzyme (3-6). Expression of UDPglucose-flavonol glucosyltransferase has been investigated in strains with unstable mutations caused by insertion of transposable controlling elements (5-10). The results of such studies suggest that controlling element mutations can alter both the structure of the enzyme and the developmental timing of gene expression. Further progress in understanding the molecular basis of such mutations is contingent on the availability of molecular probes to investigate the structure of mutant alleles. In the present communication we describe the molecular cloning of the bz locus by an indirect method involving the transposable controlling element Activator (Ac).Since the initial isolation of the white locus in Drosophila by virtue of its association with the copia transposon (11), transposon "tagging" has been used in cloning several additional Drosophila genes (12-16). The utility of transposable elements for gene isolation depends on their redundancy in the genome and the efficiency with which an insertion in the locus of interest can be identified. The isolation ofDrosophila genes by using transposon probes has been facilitated by the availability of strains with relatively few copies of a given element and by the efficient identification of clones containing the correct sequence by in situ hybridization to the correct region of salivary chromosomes (11-16).The transposable controlling elements of maize have been studied in substantial detail genetically, and mutations attributable to the insertion of well-defined elements have been identified at many maize loci (17)(18)(19)(20). The recent isolation of the Ac element and several Dissociation (Ds) elements raised the possibility of using the cloned elements to isolate other maize loci with mutations caused by these elements (21-23). The results of studies on genomic DNAs i...

The bz-mJ3CS9 allele of the bronze-1 gene in maize contains a 902-base-pair defective Suppressor-mutator (dSpm) transposable element in the second exon. Nevertheless, 40-50% of the enzymatic activity conditioned by a nonmutant allele at the bronze-1 locus is routinely recovered in crude extracts prepared from plants carrying bz-mJ3CS9 in the absence of an autonomous Suppressor-mutator element. Analyses of RNAs produced by such plants show that transcription proceeds through the dSpm. The dSpm sequence of the messenger RNA precursor is then removed by RNA splicing using the donor site of the single bronze-i intron and an acceptor site within the inverted terminal repeat of the dSpm. This results in a messenger RNA with the proper reading frame that could produce a functional enzyme. These data demonstrate that this dSpm insertion in an exon of a structural gene has produced a functional allele with a novel intron consisting, in part, of the dSpm. This mechanism appears to allow dSpm elements to reduce the impact of their insertions on gene expression.The maize defective Suppressor-mutator (dSpm) transposable elements are nonautonomous members of the Suppressor-mutator (Spm; also known as Enhancer-Inhibitor, En-I) family (1-3). They can transpose or excise only in the presence of an autonomous Spm element. One of the interesting features of dSpm elements, first noted by McClintock (2,4), is that association of these elements with structural genes in some cases gives rise to nonmutant phenotypes in the absence of Spmn. In order to understand the underlying mechanism of this phenomenon, we have analyzed an allele of this type at the maize bronze-1 locus.The bronze-1 locus is one of the many loci involved in the anthocyanin biosynthetic pathway of maize. The gene encodes UDP-glucose :flavonoid 03-D-glucosyltransferase (UFGTase, EC 2.4.1.91; refs. 5 and 6). Recessive alleles at the locus result in the bronze coloration of the aleurone layer and the brown coloration of many other plant parts as compared with the wild-type purple coloration. The mutant allele analyzed in the present study has a dSpm insertion in Bz, a nonmutant allele at the bronze-1 locus (7). This mutant allele has been designated bz-ml3CS9 (abbreviated as CS9). In the presence of an active Spm, expression of CS9 is suppressed in most cells except for infrequent revertant cell lineages. As a result, one observes small colored sectors on a bronze background. In the absence of an active Spmn, CS9 conditions full anthocyanin pigmentation.In this paper, we report that CS9 conditions a relatively high level of UFGTase activity in the absence of Spin despite the presence of the dSpm element in the second exon. Analyses of RNAs produced by CS9 in the absence of Spm show that the dSpm element contains an acceptor site for RNA splicing within its inverted terminal repeat. This enables RNA splicing to efficiently remove the dSpm sequence from the mRNA precursor, allowing for gene function.

We have sequenced genomic clones of two wild-type Bronze-1 (Bz1) alleles, and a cDNA clone from a third wild-type Bz1 allele from maize. Two overlapping transcripts initiate at least 250 bp apart. The first AUG codon after the shorter and more abundant transcript cap site(s) begins the longest open reading frame. The transcript is preceded by a putative TATA box, but not a recognizable CAAT box. The bz1 gene contains a single intron, and exhibits a strong bias for codons with the highest G+C content. Sequence polymorphisms among the Bz1 alleles include deletions/additions, a transposable element insertion, and single base pair substitutions.

Cocoa (Theobroma cacao L.) seeds are the source of chocolate flavor. The flavor develops upon post-harvest fermentation during which seed proteins are degraded. From 100 days after pollination (DAP) to maturity (160-180 DAP), three major protein bands (44, 26 and 21 kDa) are present in seed extracts subjected to denaturing polyacrylamide gel electrophoresis. The 44 and 26 kDa proteins, making up 30-50~o of total mature seed protein, behave as classical storage proteins [1], in contrast to the 21 kDa protein which increases during development but does not degrade to the same extent upon germination.Eleven percent of 20000 clones from a 130 DAP cocoa seed 2gtl0 library were positive when probed with synthetic oligonucleotides derived from a portion (residues 4-14) of the 21 kDa protein's N-terminal amino acid sequence (AlaAsn-Ser-Pro-Val-Leu-Asp-Thr-Asp-Gly-AspGlu-Leu-Gln-Thr-His-Val-Gln-Tyr-Tyr).The nucleotide and deduced amino acid sequences of an essentially full-length cDNA are shown in Figure 1. The transcript includes a 5' 78-nucleotide sequence for a 26-amino acid signal peptide which is not present at the N-terminus of the mature protein and a 3' 54-nucleotide poly(A) + tract, preceeded by two 3' AAUAAA elements. The calculated molecular weight of the mature protein (21331 Da)is similar to the sizes of protease inhibitors of the soybean trypsin inhibitor (Kunitz) class.The deduced amino acid sequence of the cocoa seed protein shows 38~o identity to a barely ct-amylase/subtilisin inhibitor (BASI [5], Fig. 2). The areas of greatest homology between the two proteins reflect areas of homology between them and two other Kunitz-type inhibitors (trypsin inhibitors of soybean [4] and winged bean [6], Fig. 2). Approximately 74~o (25 out of 34) of the residues conserved in all three of the protease inhibitors shown in Fig. 2 are also common to the cocoa protein. Considerably more identity is found among the sequences of the four proteins in the first 65 residues (35 of 65 residues of the cocoa protein matching any of the other three proteins) than in the middle or C-terminal thirds of the proteins. In addition, the highly conserved region between residues 4 and 24 (12 out of 24) is also highly conserved between at least four other Kunitz-type protease inhibitors from seeds of leguminous plants [3]. Four cysteine residues strictly conserved among the amino acid sequences of the protease inhibitors, and believed to be involved in disulfide bonding in BASI, are also conserved in the cocoa seed protein. The protein also shows much similarity (34~o identity) to sporamin b [2] of sweet potato tubers (Fig. 2). Many areas of high homology between the two The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number X54509 Fig. 1. Nucleotide and deduced amino acid sequence of a cDNA clone encoding the 21 kDa cocoa seed protein. The arrow indicates the probable cleavage site of the putative 26-amino acid signal polypeptide from the mature protein. The u...

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