Characterization of a maize G-box binding factor that is induced by hypoxia

G-box cis-acting DNA sequence elements are present in the promoter region of a number of signal-inducible plant genes. In many cases this motif is essential for gene expression. Maize nuclear extracts contain a protein complex that binds specifically to the G-box sequence. Previously, a protein called GF14 was described that is physically associated with the G-box binding complex, but is not a DNA-binding factor in and of itself. This paper reports the isolation of a cDNA encoding a maize G-box binding factor (GBF). The deduced amino acid sequence indicates that maize GBF1 is a basic region-leucine zipper protein. GBF1 binds to the G-box element with specificity similar to that of the binding activity in nuclear extracts. Furthermore, maize GBF1 and the factor detected in nuclear extract are identical in their molecular weight and are immunologically related. GBF1 mRNA accumulates rapidly in hypoxically induced maize cells prior to the increase in Adh1 mRNA levels. Taken together with results that indicate that GBF1 binds to the hypoxia-responsive promoter of maize Adh1, these observations suggest that GBF1 may be one of the factors involved in the activation of Adh1.     

Functional analysis of the proteasome regulatory particle

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from rpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.     

Functional analysis of regulatory single-nucleotide polymorphisms

PURPOSE OF REVIEW: The identification of regulatory polymorphisms has become a key problem in human genetics. In the past few years there has been a conceptual change in the way in which regulatory single-nucleotide polymorphisms are studied. We revise the new approaches and discuss how gene expression studies can contribute to a better knowledge of the genetics of common diseases. RECENT FINDINGS: New techniques for the association of single-nucleotide polymorphisms with changes in gene expression have been recently developed. This, together with a more comprehensive use of the old in-vitro methods, has produced a great amount of genetic information. When added to current databases, it will help to design better tools for the detection of regulatory single-nucleotide polymorphisms. SUMMARY: The identification of functional regulatory single-nucleotide polymorphisms cannot be done by the simple inspection of DNA sequence. In-vivo techniques, based on primer-extension, and the more recently developed 'haploChIP' allow the association of gene variants to changes in gene expression. Gene expression analysis by conventional in-vitro techniques is the only way to identify the functional consequences of regulatory single-nucleotide polymorphisms. The amount of information produced in the last few years will help to refine the tools for the future analysis of regulatory gene variants.     

DNA binding activity of the Arabidopsis G-box binding factor GBF1 is stimulated by phosphorylation by casein kinase II from broccoli.

To study the phosphorylation of one of the G-box binding factors from Arabidopsis (GBF1), we have obtained large amounts of this protein by expression in Escherichia coli. Bacterial GBF1 was shown to be phosphorylated very efficiently by nuclear extracts from broccoli. The phosphorylation activity was partially purified by chromatography on heparin-Sepharose and DEAE-cellulose and was characterized. It showed the essential features of casein kinase II activity: utilization of GTP in addition to ATP as a phosphate donor, strong inhibition by heparin, preference for acidic protein substrates, salt-induced binding to phosphocellulose, and salt-dependent deaggregation. The very low Km value for GBF1 (220 nM compared to approximately 10 microM for casein) was in the range observed for identified physiological substrates of casein kinase II. Phosphorylation of GBF1 resulted in stimulation of the G-box binding activity and formation of a slower migrating protein-DNA complex. The conditions of this stimulatory reaction fully corresponded to the properties of casein kinase II, in particular its dependence on the known phosphate donors. The DNA binding activity of the endogenous plant GBF was shown to be reduced by treatment with calf alkaline phosphatase; this reduction was diminished by addition of fluoride and phosphate or incubation in the presence of casein kinase II and ATP.     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel