Biomedical subjects

J G Tokuhisa

Publications and source records attributed to J G Tokuhisa.

4 recordsLinked to original sources

PubMed1990

A DNA-binding protein factor recognizes two binding domains within the octopine synthase enhancer element.

A protein that binds to the enhancing element of the octopine synthase gene has been identified in nuclear extracts from maize cell suspension cultures. Two protein-DNA complexes are distinguishable by electrophoretic mobility in gel retardation assays. Footprint analyses of these low and high molecular weight complexes show, respectively, half and complete protection of the ocs-element DNA from cleavage by methidiumpropyl-EDTA.FE(II). Two lines of evidence indicate that the element has two recognition sites, each of which can bind identical protein units. Elements that are mutated in one or the other half and form only the low molecular weight complex interfere with the formation of both the low and high molecular weight complexes by the wild-type element. Protein isolated from a complex with only one binding site occupied can bind to the wild-type ocs-element and generate complexes with protein occupying one or both binding sites. Occupation of both sites of the ocs-element is a prerequisite for transcriptional enhancement.

Amino Acid Oxidoreductases↗

PubMed1990

OCSBF-1, a maize ocs enhancer binding factor: isolation and expression during development.

The ocs-elements comprise a family of related 20-base pair DNA sequences with dyad symmetry that are functional components of the promoters of several genes introduced into the plant nucleus by Agrobacterium transformation or infection by DNA viruses. We describe the isolation and characterization of a maize cDNA that encodes a protein, OCSBF-1, that binds specifically to ocs-element sequences. The 21-kilodalton OCSBF-1 protein was encoded by a single copy, intron-less gene. The gene was differentially expressed in maize plants. Developing leaves had a gradient of OCSBF-1 mRNA with the basal portion of the leaves, which contain dividing and differentiating cells, having 40-fold to 50-fold higher levels of OCSBF-1 transcripts than the apical portion of the leaves, where the cells are fully differentiated. Roots and shoots of young plants had levels of OCSBF-1 mRNA similar to the basal portions of developing leaves. OCSBF-1 contained a small basic amino acid region and a potential leucine zipper motif homologous to the DNA-binding domains of the basic region-leucine zipper family of transcription factors such as Jun and GCN4. A truncated protein with the amino-terminal 76 amino acids of OCSBF-1, encompassing the basic domain and leucine zipper motif, still bound to ocs-element sequences in vitro. OCSBF-1 was able to bind to a site within each half of the ocs-element as well as to animal AP-1 and CREB sites.

Amino Acid Sequence↗

PubMed1989

The ocs-element is a component of the promoters of several T-DNA and plant viral genes.

The ocs-element is an enhancer element first identified in the promoter of the octopine synthase gene (OCS) where it occurs as a 16 bp palindromic sequence. The transcriptional enhancing activity of the ocs-element correlated with in vitro binding of a transcription factor. We have now identified ocs-elements in the promoter regions of six other T-DNA genes involved in opine synthesis and three plant viral promoters including the 35S promoter of cauliflower mosaic virus. These elements bind the ocs transcription factor in vitro and enhance transcription in plant cells. Comparison of the sequences of these 10 elements has defined a 20 bp consensus sequence, TGACG(T/C)AAG(C/G)(G/A)(A/C)T(G/T)ACG(T/C)(A/C)(A/C), which includes the 16 bp palindrome in its central region. We propose the name ocs-element for this class of promoter elements of similar sequence and function.

Base Sequence↗

PubMed1989

Saturation mutagenesis of the octopine synthase enhancer: correlation of mutant phenotypes with binding of a nuclear protein factor.

A 16-base-pair palindrome from the Agrobacterium tumefaciens octopine synthase gene functions as a constitutive enhancer in plant protoplasts. Degenerate oligonucleotide mutagenesis provided single base substitutions at every position in the element and a number of multiple base substitutions. The effects of these changes were determined in transient expression assays with tobacco and maize protoplasts. The majority of single and double base changes had little effect on the activity of the octopine synthase enhancer, but nearly all mutants with more than two base changes had low to essentially no activity. There were five positions where particular single base changes resulted in a 4- to 10-fold loss in enhancer activity. The distribution of these positions within the palindrome was asymmetric. Single base deletions had essentially no activity, demonstrating that the octopine synthase enhancer cannot tolerate internal changes in spacing. We find a strong correlation between mutant phenotype and reduced binding of a protein factor, suggesting that the DNA-protein complex is responsible for the transcriptional enhancement; the functionally active form of the DNA-protein complex probably involves more than a single protein molecule. The mutants exhibit similar phenotypes in protoplasts of both tobacco and maize, implying conservation of the DNA-protein interactions of the ocs enhancer sequence in monocotyledonous and dicotyledonous plants.

Amino Acid Oxidoreductases↗