Interaction of trans and cis regulatory elements in the INO1 promoter of Saccharomyces cerevisiae.

Electrophoretic mobility shift assays (EMSA) were used to define the regions of the INO1 promoter that interact with factors present in extracts prepared from the yeast, Saccharomyces cerevisae. These experiments identified three different types of protein:DNA complexes that assemble with the INO1 promoter. Formation of one type of complex depended on functional alleles of previously described regulatory genes, INO2 and INO4, that encode positive regulatory factors. Formation of the INO2/INO4-dependent complexes was increased when extracts prepared from cells grown under derepressing conditions (i.e. absence of inositol and choline). A second type of complex was dependent on the OPI1 gene, that encodes a negative regulator. Computer-aided sequence analysis of the promoters of several genes encoding phospholipid biosynthetic enzymes revealed a highly conserved nine basepair element (5'-ATGTGAAAT-3'), designated 'nonamer' motif, that is similar to the octamer motif identified in the promoters of mammalian immunoglobulin genes. The nonamer motif was shown to form a specific complex with a factor, designated nonamer binding factor (NBF). Extracts prepared from Schizosaccharomyces pombe formed a nonamer-specific complex.     

Characterization of a novel class of plant homeodomain proteins that bind to the C4 phosphoenolpyruvate carboxylase gene of Flaveria trinervia

We are interested in the regulatory mechanisms responsible for the mesophyll-specific expression of C₄ phosphoenolpyruvate carboxylase (PEPCase). A one-hybrid screen resulted in the cloning of four different members of a novel class of plant homeodomain proteins, which are most likely involved in the mesophyll-specific expression of the C₄ PEPCase gene in C₄ species of the genus Flaveria. Inspection of the homeodomains of the four proteins reveals that they share many common features with homeodomains described so far, but there are also significant differences. Interestingly, this class of homeodomain proteins occurs also in Arabidopsis thaliana and other C₃ plants. One-hybrid experiments as well as in vitro DNA binding studies confirmed that these novel homeodomain proteins specifically interact with the proximal region of the C₄ PEPCase gene. The N-terminal domains of the homeodomain proteins contain highly conserved sequence motifs. Two-hybrid experiments show that these motifs are sufficient to confer homo- or heterodimer formation between the proteins. Mutagenesis of conserved cysteine residues within the dimerization domain indicates that these residues are essential for dimer formation. Therefore, we designate this novel class of homeobox proteins ZF-HD, for zinc finger homeodomain protein. Our data suggest that the ZF-HD class of homeodomain proteins may be involved in the establishment of the characteristic expression pattern of the C₄ PEPCase gene.     

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.     

In silico characterization of the INO80 subfamily of SWI2/SNF2 chromatin remodeling proteins

Proteins belonging to SNF2 family of DNA dependent ATPases are important members of the chromatin remodeling complexes that are implicated in epigenetic control of gene expression. The yeast Ino80, the catalytic ATPase subunit of the INO80 complex, is the most recently described member of the SNF2 family. Outside the conserved ATPase domain, it has very little similarity with other well-characterized SNF2 proteins hence it is believed to represent a new subfamily. We have identified new members of this subfamily in different organisms and have detected characteristic features of this subfamily. Using various data mining tools we have identified a new, previously undetected domain in all members of this subfamily. This domain designated DBINO is characteristic of the INO80 subfamily and is predicted to have DNA-binding function. The presence of this domain in all the INO80 subfamily proteins from different organisms suggests its conserved function in evolution.