The activation domain of the maize transcription factor Opaque-2 resides in a single acidic region.

The maize (Zea mays L.) endosperm specific transcription factor, encoded by the Opaque-2(O2) locus, functions in vivo to activate transcription from its target promoters.O2 regulates the expression of a major storage protein class, the 22 kDa zeins, and of a type I ribosome inactivating protein, b-32, during maturation phase endosperm development. The coding sequence of O2, which indicates it to be a member of the basic region-leucine zipper (bZIP) class of DNA-binding proteins, contains a number of regions rich in either proline or acidic residues which are candidates for activation domains. In functional assays using tobacco mesophyll protoplasts, the level of transactivation conferred by a series of O2-deletion constructs was tested using as a reporter a fusion of the b-32 target promoter to beta-glucuronidase (GUS). The results indicate that O2 has a single acidic activation domain, located near the N-terminus of the protein (amino acids 41-91). The ability of a shorter part of this domain (amino acids 39-82) to confer transactivation was also demonstrated in domain swapping experiments, using fusions of the O2 polypeptide sequence to the DNA-binding domain of the parsley (Petroselinum crispum) transcription factor CPRF1.

The role of multiple binding sites in the activation of zein gene expression by Opaque-2.

Opaque-2 (O2) encodes a transcriptional activator of the basic domain-leucine zipper (bZIP) class, which controls the expression level in maize endosperm of the 22kD alpha-zeins and a number of non-storage proteins. The interaction of the O2 protein at three clustered binding sites on an isolated 22 kD zein gene promoter has been investigated. O2 is shown to transactivate transcription from these sites in tobacco mesophyll protoplasts as well as in maize endosperm cells transformed by particle bombardment. The binding sites have been mutated by base exchanges, singly or in different combinations, to determine their contribution to transactivation in vivo in both the leaf protoplast and the maize endosperm system. The effect of these mutations on binding of O2 in vitro was determined by electrophoretic mobility shift assays (EMSA), using O2 protein expressed in E. coli. Two of the sites seemed to be equally effective in responding to Opaque-2 in vivo in both cell types, although one of them does not contain an ACGT core sequence, and has a lower affinity for O2 in vitro than the ACGT-containing binding site. A third site, which has the lowest affinity of all three, confers no detectable O2-dependent promoter activation alone, but significantly increases activation in combination with either one of the other sites. Hence, weaker O2 binding sites can still mediate major O2-dependent effects when present in target promoters in vivo.

The transcriptional activator Opaque-2 controls the expression of a cytosolic form of pyruvate orthophosphate dikinase-1 in maize endosperms.

The maize Opaque-2 (O2) protein is a transcription factor of the basic/leucine-zipper class, involved in the regulation of endosperm proteins including the 22kDa alpha-zein storage proteins and b32 protein. In this study we have focussed our attention on the relationship between O2 and the cyPPDK1 gene, which encodes a cytoplasmic pyruvate orthophosphate dikinase (PPDK) isoform. The results of this study showed that PPDK activity is detectable in wild-type maize endosperms, while in o2 mutant endosperms, the levels of PPDK protein, mRNA and enzymatic activity are reduced, indicating that O2 is involved in the regulation of cyPPDK1 in this tissue. By employing transient expression experiments in tobacco mesophyll protoplasts, we have demonstrated that the O2 protein can activate expression of a chloramphenicol acetyl transferase reporter gene placed under the control of the cyPPDK1 promoter. An in vitro binding assay and DNaseI footprint analysis demonstrated that a specific sequence in the cyPPDK1 promoter can be recognized and protected by maize O2 protein. The regulation by the O2 locus of cyPPDK1 reported here, and control of alpha-zein synthesis by O2 suggest that the O2 protein may play a more general role in maize endosperm development than previously thought.

A 10-bp deletion in the apolipoprotein epsilon gene causing apolipoprotein E deficiency and severe type III hyperlipoproteinemia.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2. We identified a 30-year-old male German of Hungarian ancestry with severe type III HLP and apo E deficiency. The disease was expressed in an extreme phenotype with multiple cutaneous xanthomas. Apo E was detectable only in trace amounts in plasma but not in the different lipoprotein fractions. Direct sequencing of PCR-amplified segments of the apo epsilon gene identified a 10-bp deletion in exon 4 (bp 4037-4046 coding for amino acids 209-212 of the mature protein). The mutation is predictive for a reading frameshift introducing a premature stop codon (TGA) at amino acid 229. By western blot analysis, we found small amounts of a truncated apo E in the patient's plasma. Family analysis revealed that the proband was homozygous--and 10 of 24 relatives were heterozygous--for the mutation. Heterozygotes had, as compared to unaffected family members, significantly higher triglycerides (TG), very low-density lipoprotein (VLDL) cholesterol and a significantly higher VLDL cholesterol-to-serum TG ratio, which is indicative of a delayed remnant catabolism. We propose that the absence of a functionally active apo E is the cause of the severe type III HLP in the patient and that the mutation, even in a single dose in heterozygotes, predisposes in variable severity to the phenotypic expression of the disease.

Functional expression of the transcriptional activator Opaque-2 of Zea mays in transformed yeast.

The aim of this research was to determine whether the structural homology between the O2 gene, a maize transcriptional activator, and the GCN4 gene, a yeast transcriptional factor, is reflected at the level of function. The O2 cDNA was cloned in the yeast expression vector pEMBLyex4 under the control of a hybrid inducible promoter, and used to transform the yeast Saccharomyces cerevisiae. Transformed yeast cells produced O2 mRNA and a polypeptide immunoreactive with anti-O2 antibodies during growth in galactose. The heterologous protein was correctly translocated into the yeast nuclei, as demonstrated by immunofluorescence, indicating that the nuclear targeting sequences of maize are recognized by yeast cells. Further experiments demonstrated the ability of O2 to rescue a gcn4 mutant grown in the presence of aminotriazole, an inhibitor of the HIS3 gene product, suggesting that O2 activates the HIS3 gene, gene normally under control of GCN4. It was shown that the O2 protein is able to trans-activate the HIS4 promoter in yeast cells and binds to it in vitro. The sequence protected by O2, TGACTC, is also the binding site for GCN4. Finally, the expression of O2 protein in yeast did not produce alterations during batch growth at 30 degrees C, while transformants expressing O2 protein showed a conditionally lethal phenotype when grown in galactose at 36 degrees C; this phenotype mimics the behaviour of gcd mutants. The results support the idea that basic mechanisms of transcription control have been highly conserved in eukaryotes.

Translation of the mRNA of the maize transcriptional activator Opaque-2 is inhibited by upstream open reading frames present in the leader sequence.

The protein encoded by the Opaque-2 (O2) gene is a transcription factor, translated from an mRNA that possesses an unusually long 5' leader sequence containing three upstream open reading frames (uORFs). The efficiency of translation of O2 mRNA has been tested in vivo by a transient assay in which the level of activation of the b32 promoter, a natural target of O2 protein, is measured. We show that uORF-less O2 alleles possess a higher transactivation value than the wild-type allele and that the reduction in transactivation due to the uORFs is a cis-dominant effect. The data presented indicate that both uORF1 and uORF2 are involved in the reducing effect and suggest that both are likely to be translated.

The maize regulatory locus Opaque-2 encodes a DNA-binding protein which activates the transcription of the b-32 gene.

The maize locus, Opaque-2, controls the expression in developing endosperm of structural genes encoding a family of storage proteins, the 22 kd zeins, and an abundant albumin, termed b-32. It is shown that the promoter of the b-32 gene is activated in vivo in the presence of the O2 gene product and that the information necessary for this activation resides in a 440 bp DNA fragment containing five O2 binding sites (GATGAPyPuTGPu). Two of these sites are embedded in copies of the 'endosperm box', a motif thought to be involved in endosperm-specific expression, which is also represented in 22 kd zein promoters. The O2 protein is also shown to be capable of binding in vitro and activating in vivo, its own promoter.