Preliminary tests have shown that the endosperms of maize seeds homozygous for the opaque-2 mutant gene have a higher lysine content than normal kernels. As a critical test, a backcross progeny was divided into opaque-2 and normal kernels, the endosperms separated, and the amino acids determined. The opaque-2 endosperms had a different amino acid pattern and 69 percent more lysine than the normal seeds. The major reason for these changes is the synthesis of proteins with a greater content of basic amino acids in the acid-soluble fraction of the mutant endosperm. This is accompanied by a reduction in the ratio of zein to glutelin.

Evidence is presented to show that the Sh locus specifies sucrose synthetase in the developing endosperm of maize. The sh/sh/sh endosperm possesses less than 10% sucrose synthetase activity as compared to the normal Sh/sh/sh endosperm. The residual enzyme activity in five independently derived mutant genotypes is attributable to a protein molecule of different electrophoretic and immunochemical specificities that is presumably independent of the sh locus. Sucrose synthetase activity in the embryo in both the genotypes is electrophoretically indistinguishable from the one present in the mutant endosperm. Mutant endosperm has a reduced starch content as compared to the normal. This observation constitutes genetic evidence supporting a critical role for sucrose synthetase in starch biosynthesis.

Many of the sugary-i mutants of maize (Zea mays L.) have the highly branched water-soluble polysaccharide, phytoglycogen, in quantities equal to or greater than starch as an endosperm storage product in mature seeds. We find that all sugary mutants investigated are deficient in debranching enzyme la-(1, 6)-glucosidasel activity in endosperm tissue 23 days postpollination and suggest that this deficiency is the primary biochemical lesion leading to phytoglycogen accumulation in sugary endosperms. This would indicate that the amylopectin component of starch depends on an equilibrium between the activities of branching enzymes introducing a-1,6 branch points into the linear a-1,4 glucans and debranching enzymes. The debranching enzyme activities from nonsugary endosperms can be separated into three peaks on a hydroxyapatite column. The sugary endosperm extracts lack one ofthese peaks ofactivity while the other two fractions have much reduced activity. The embryos of developing seeds (23 days after pollination) from both sugary and nonsugary genotypes have equivalent debranching activity. The debranching enzyme activity of developing endosperms is proportional to the number of copies (O to 3) of the nonmutant (Su) allele present suggesting that the Su allele may be the structural gene for this debranching enzyme, although this is not definitive. This identification of debranching enzyme activity as being the biochemical lesion in sugary endosperms is consistent with several previous observations on the mutant.The sugary-l (sul) mutant of maize, Zea mays L., which is the usual sweet corn of commerce, has been known and utilized since preColumbian times (21), and specific efforts to improve particular varieties of sweet corn date back to the middle of the nineteenth century (18). In spite of this lengthy association with man, the specific biochemical lesion induced by mutations at the su locus has not been known. Sumner and Somers (20) reported that the principal polysaccharide storage product in su endosperms was not starch, but a highly branched, water-soluble polysaccharide of high mol wt which Sumner and Somers referred to as phytoglycogen. Erlander ( 11) MATERIALS AND METHODS Biological Materials. Self-pollinated seeds ofthe maize inbred W64A or the hybrid W64A x 182E were used as the nonsugary controls. The first group of sugary mutants examined contained the reference allele at the su locus, su-R, and an intermediate allele su-Bn2. These alleles had been placed in the W64A genotype by repeated backcrosses. A second group of sugary alleles contained su-8115, su-8116, su-8117, and su-8135. These were presumed to have been independently induced by ethylmethanesulfonate (EMS) treatment since they were isolated in the progeny of inbred W22 plants exposed to the mutagen. For simplicity, we refer to su endosperms throughout the report. It should be understood that this means homozygous sugary (sul su/su) endosperms.

The mutant floury-2 results in the production of maize endosperm proteins with an altered amino acid pattern. The lysine concentration is high, eapproximately equal to that in mutant opaque-2, and the methionine concentration is higher than in any other stock tested. Other mutants of similar phenotype!, opaque-1, floury-1, and soft-starch do not cause major changes in amino acid Pattern.

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...