Molecular analysis of the maize anthocyanin regulatory locus C1

The C1 gene of maize plays a regulatory role in the production of anthocyanin pigments in the aleurone layer of the endosperm. As an initial step toward understanding the molecular details of how C1 controls pigment biosynthesis, we cloned the C1 gene. This was accomplished by first cloning a mutable allele of C1, c1-m5, which contains the transposable element Spm. A combination of molecular and genetic analysis was used to identify the Spm at the C1 locus. Individual genomic DNAs from a population in which the c1-mutable phenotype was segregating with the recessive c1 phenotype were digested with methyl-sensitive restriction enzymes and probed with a small DNA fragment derived from a defective Spm. One Sal I restriction fragment complementary to the Spm probe was shown to be present in the DNA of individuals with the c1-m5 phenotype but absent from DNA of individuals with a recessive c1 phenotype. Subsequent cloning and restriction analysis of this fragment revealed sequences flanking the Spm that proved to be C1-specific. A DNA fragment derived from the flanking sequences was then used as a probe to clone the wild-type C1 gene and several additional alleles of C1, including one stable recessive, two mutations caused by Ds insertions, one mutation induced by insertion of a defective Spm, and two dominant mutations, C1-S and C1-I. RNA blot hybridization analysis of three C1 alleles indicates that C1 regulation of the Bz1 and A1 structural genes in the anthocyanin biosynthetic pathway is at the transcriptional level.