The opaque-2 locus (o2) in maize regulates the expression of many members of the zein multigene family of storage proteins. cDNA clones for a wild-type allele of the (o2) locus (02) were isolated from a maize endosperm cDNA library and sequenced. We found a 258-nucleotide 5' leader sequence containing three short open reading frames followed by a sequence specifying a protein of 437 amino acids. The presumptive amino acid sequence of the protein (02) specified by the 02 cDNA contains a "leucine-zipper" domain characteristic of some mammalian and fungal transcription activation factors. lacZ-02 fusion constructs, using nearly the entire coding region of 02 or only a fragment specifying the leucinezipper domain, were expressed inEscherichia coli. In an in vitro binding assay, the (3-galactosidase-02 fusion proteins bound to two specific regions on the 5' side of the coding sequence in a zein genomic clone. This suggests that the 02 protein affects iein transcription through direct interaction with one or more zein promoter elements.The opaque-2 mutation conditions a phenotypic change from hard glassy kernels to seeds with a soft powdery endosperm that are opaque to light. This change is correlated with a significant reduction in the zein storage proteins (1, 2). In mature wild-type kernels, the zeins constitute about 60% ofthe total endosperm protein (1). Zeins can be separated on SDS/ polyacrylamide gels into several size classes based on their apparent molecular weights. The two major classes ofthe zein family, the 19-and 22-kDa zeins, can be resolved by isoelectric focusing-SDS/gel electrophoresis into 15-22 polypeptides (3,4). This charge heterogeneity reflects polymorphisms within different zein structural genes (5, 6). The o2 mutation causes a suppression of specific members, most generally of the 22-kDa class (5, 7, 8), resulting in an overall reduction of 50-70% in zein proteins (1, 2). This is apparently due to a lower rate of zein transcription, particularly those encoding the 22-kDa class (9). We would like to understand how the o2 locus differentially promotes the transcription of specific members of a gene family in which synthesis is coordinately regulated (10). To this end, we isolated a wild-type allele of the o2 locus (02) by transposon tagging (11). Probes of the gene were used to show the tissue-specific expression of o2 and its independence from two other regulators of zein synthesis, o7 and f12 (11). The latter results have been confirmed in similar analyses performed by others (12).Here we report the isolation and the sequence of a cDNA clone of the o2 locus. § By comparing the deduced 02 protein sequence with, a protein data base, we found that the 02 protein shares sequence similarity with the "leucine-zipper" DNA binding domain characteristic of several mammalian oncogenes and fungal transcription activation factors (13,14). By DNA binding studies we demonstrated that the 02 protein or a fragment containing the leucine-zipper domain binds specifically to a genomic zein clone, the t...

The Cl 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 Cl controls pigment biosynthesis, we cloned the Cl gene. This was accomplished by first cloning a mutable allele of Cl, cl-mS, which contains the transposable element Spm. A combination of molecular and genetic analysis was used to identify the Spm at the Cl locus. Individual genomic DNAs from a population in which the cl-mutable phenotype was segregating with the recessive cl 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 cl-m5 phenotype but absent from DNA of individuals with a recessive cl phenotype. Subsequent cloning and restriction analysis of this fragment revealed sequences flanking the Spm that proved to be Cl-specific. A DNA fragment derived from the flanking sequences was then used as a probe to clone the wild-type Cl gene and several additional alleles of Cl, including one stable recessive, two mutations caused by Ds insertions, one mutation induced by insertion of a defective Spm, and two dominant mutations, Cl-S and C1-. RNA blot hybridization analysis of three Cl alleles indicates that Cl regulation of the Bzl and Al structural genes in the anthocyanin biosynthetic pathway is at the transcriptional level.

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Genetic analyses suggested that the opaque-2 (o2) locus in maize acts as a positive, transacting, transcriptional activator of the zein seed storage-protein genes. Because isolation of the gene is requisite to understanding the molecular details of this regulation, transposon mutagenesis with the transposable element suppressor-mutator (Spm) was carried out, and three mutable o2 alleles were obtained. One of these alleles contained an 8.3-kilobase autonomous Spm, another a 6.8-kilobase nonautonomous Spm, and the third an unidentified transposon that is unrelated to Spm. A DNA sequence flanking the autonomous Spm insertion was verified to be o2-specific and provided a probe to clone a wild-type allele. Northern blots indicated that the gene is expressed in wild-type endosperm but not in leaf tissues or in endosperms homozygous for a mutant allele of the O2 gene. A transcript was detected in endosperms homozygous for mutations at opaque-7 and floury-2, an indication that O2 expression is independent of these two other putative regulators of zein synthesis.

We present here the annotation of the complete genome of rice Oryza sativa L. ssp. japonica cultivar Nipponbare. All functional annotations for proteins and non-protein-coding RNA (npRNA) candidates were manually curated. Functions were identified or inferred in 19,969 (70%) of the proteins, and 131 possible npRNAs (including 58 antisense transcripts) were found. Almost 5000 annotated protein-coding genes were found to be disrupted in insertional mutant lines, which will accelerate future experimental validation of the annotations. The rice loci were determined by using cDNA sequences obtained from rice and other representative cereals. Our conservative estimate based on these loci and an extrapolation suggested that the gene number of rice is ∼32,000, which is smaller than previous estimates. We conducted comparative analyses between rice and Arabidopsis thaliana and found that both genomes possessed several lineage-specific genes, which might account for the observed differences between these species, while they had similar sets of predicted functional domains among the protein sequences. A system to control translational efficiency seems to be conserved across large evolutionary distances. Moreover, the evolutionary process of protein-coding genes was examined. Our results suggest that natural selection may have played a role for duplicated genes in both species, so that duplication was suppressed or favored in a manner that depended on the function of a gene.