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R J Ferl

Publications and source records attributed to R J Ferl.

At least 55 records · Page 3Linked to original sources
PubMed1994

In vivo footprinting identifies an activating element of the maize Adh2 promoter specific for root and vascular tissues.

In vivo footprinting identifies four putative cis elements of Adh2 that interact with protein factors within the DNase I hypersensitive domains of the 5' flanking region. The power of in vivo footprinting to identify functionally significant sites within a gene promoter was tested by biochemical and transgenic analyses of the putative element at position -160. Biochemical analyses show that proteins isolated from maize cell suspensions will bind to the Adh2 promoter in vitro to generate a footprint at -160 identical to that seen in vivo. The partially purified factor bound to the promoter in vitro can be specifically competed with fragments of DNA containing the element sequence, further demonstrating that a specific protein generates the footprint over that sequence. Transgenic analyses indicate that the -160 element is a functional element of the maize Adh2 promoter that acts as an activator in the meristem and vascular tissue of roots and in the vascular tissue of stems and leaves.

Alcohol Dehydrogenase↗
PubMed1994

Two genes encoding GF14 (14-3-3) proteins in Zea mays. Structure, expression, and potential regulation by the G-box binding complex.

Two maize (Zea mays) genes, designated GRF1 and GRF2, have been isolated and characterized. The proteins encoded by these genes, called GF14 proteins, participate in protein/DNA complexes and show more than 60% identity with a highly conserved, widely distributed protein family, collectively referred to as 14-3-3 proteins. Members of the 14-3-3 protein family have been reported to activate Tyr and Trp hydroxylases, modulate protein kinase C activity, and activate ADP-ribosyltransferase. The mRNAs of the GRF genes are encoded by six exons interrupted by five introns. The transcriptional units of the GRF genes were found to be very similar, with complete conservation of the intron positions. In addition, the length and nucleotide sequences of the two genes' introns were highly conserved. The 5' flanking sequences of the two GRF genes were compared and regions of homology and divergence identified. This comparison revealed the presence of a conserved G-box element in the 5' flanking region of both genes. Electrophoretic mobility shift assays of maize protein extract with the GRF G-box indicates that GBF binds to this G-box site in the 5' up stream region of GRF. Antibody supershifts indicate that GF14 protein is associated with the G-box-binding complex that interacts with the GRF upstream region.

14-3-3 Proteins↗
PubMed1994

Structural features of the maize sus1 gene and protein.

Genomic clones, cDNA clones, and protein of the maize (Zea mays L.) Suc synthase1 (sus1) gene were isolated and sequenced. Termini (5' and 3') of the transcribed unit were identified. The SUS1 protein was purified from tissue culture cells as a phosphorylated protein. The overall structure of sus1 is virtually identical with that of the paralogous gene, shrunken1 (sh1); however, the last intron of sh1 is missing in sus1. This intron bears much sequence similarity with the adjacent exon, suggesting that the intron arose from an internal duplication. Although the placement of the other 14 introns is identical in both genes, the introns exhibit markedly greater differences in size and sequence relative to that shown by the exons. An explanation for the differential rate of divergence of exons and introns is selection pressure for gene function. Additionally, comparisons of coding regions of plant sucrose synthases show that sh1-like and sus1-like genes can be found in all monocots so far analyzed. These latter observations point to an important role played by both genes in this group of plants.

Base Sequence↗
PubMed1994

Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog.

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.

Amino Acid Sequence↗
PubMed1993

Gene sequence tags from Plasmodium falciparum genomic DNA fragments prepared by the "genease" activity of mung bean nuclease.

A genes-first approach to genome sequencing is described which efficiently generates gene sequence tags from genomic DNA. Mung bean nuclease (EC 3.1.30.1) cleaves the genomic DNA of many organisms before and after genes and within some introns. Analysis of gene sequence tags prepared from mung bean nuclease-digested Plasmodium falciparum DNA demonstrates that this method has several advantages over the popular cDNA expressed sequence tag approach. To date, 673 sequence tags containing over 215 kb of sequence have been generated from 400 clones. Sixty clones (15%) have significant similarity to sequences in the protein and translated nucleic acid data bases. These represent 51 unique genes, of which only 5 encode previously known P. falciparum proteins. The identified proteins include those expressed in erythrocytic, exoerythrocytic, and gametocytic stages of the parasite. Thirty percent of clones identified appear to carry complete coding regions. The spacer DNA separating genes is rarely cloned. These gene sequence tags will form a useful data base from which to initiate projects to develop new therapeutics, vaccines, and strategies to control human malaria.

Animals↗
PubMed1993

Osmium tetroxide footprinting of a scaffold attachment region in the maize Adh1 promoter.

Osmium tetroxide (OsO4) reacts with the thymine residues of double-stranded DNA, but thymines that are unpaired or under torsional stress are hyperreactive. Although OsO4 hyperreactivity has been primarily utilized to identify Z-DNA structures in supercoiled plasmids, OsO4 will also identify other torsional perturbations of DNA. In this study, OsO4 was used to footprint an AT-rich region (between -780 and -500) of the maize Adh1 promoter. Hyperreactive sites were identified both in vitro and in vivo in an area that coincides with AT motifs similar to those found in scaffold attachment regions. Further, the region of OsO4 hyperreactivity lies within a fragment of DNA that is associated with the nuclear scaffold in histone-depleted nuclei.

Alcohol Dehydrogenase↗
PubMed1993

Homopurine/homopyrimidine sequences as potential regulatory elements in eukaryotic cells.

1. Homopurine/homopyrimidine (PuPy) repetitive duplex sequences can form intramolecular triplexes (H-DNA) or intermolecular triplexes with a third strand in a sequence-specific manner. 2. Such sequences are present in natural genomes within 5'- and 3'-flanking sequences and coding regions of genes. Triplex DNA structures have been detected in vitro and in vivo and have been immunolocalized to chromosomes by triplex-specific monoclonal antibody approaches. 3. Intermolecular triplex formation represses gene expression at the transcriptional level and is also useful in genomic mapping, gene cloning, sequence-specific drug delivery, and selective modulation of gene expression.

Animals↗
PubMed1992

Brain proteins in plants: an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding complex.

The G box is a well-characterized cis-acting DNA regulatory element found in the promoters of several seemingly unrelated plant genes, including the alcohol dehydrogenase (Adh) gene of Arabidopsis thaliana. Using a monoclonal antibody screening approach coupled with electrophoretic mobility shift assays, we have isolated a cDNA clone encoding a protein that is part of the in vitro protein/G box complex. The derived amino acid sequence is homologous to a class of proteins in mammalian brains described as protein kinase C inhibitors and as activators of tyrosine and tryptophan hydroxylases, the rate-limiting enzymes in the pathways leading to the catecholamines and serotonin. The fact that a homologous member of this regulatory protein family is found in plants and is associated with binding to transcriptional regulatory elements suggests a much wider role for these proteins.

Amino Acid Sequence↗
PubMed1992

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo.

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from -44 to -79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.

Alcohol Dehydrogenase↗
PubMed1992

Functional elements of the Arabidopsis Adh promoter include the G-box.

The alcohol dehydrogenase (Adh) gene of Arabidopsis is expressed constitutively in immature seedlings and cells in suspension, and may be induced by hypoxic stress only in roots of mature plants. Deletions and G-box mutations of the Adh promoter were assayed in Arabidopsis protoplasts by PEG-mediated transient expression. Sequence domains necessary for full gene activity are confined to the 384 bp immediately 5' to the transcription start site, and deletion to -177 results in greater than 90% reduction in promoter activity. Site-specific mutations of G-box bases result in greater than 60% reduction in activity and disrupt G-box factor binding in vitro.

Alcohol Dehydrogenase↗
PubMed1992

Chemical detection of Z-DNA within the maize Adh1 promoter.

Z-DNA is a left-handed helix which can form within tracts of alternating purines and pyrimidines. Tracts of potential Z-DNA identified by sequence inspection are often noted within regulatory portions of genes, but evidence that these tracts of sequence actually exist as Z-DNA is very limited, and not available for any plant gene. In this study, the chemical probes osmium tetroxide, diethylpyrocarbonate and hydroxylamine were used to show that a tract of alternating purines and pyrimidines in the Adh1 promoter (from -311 to -325) actually assumes a Z-DNA conformation under superhelical stress in vitro.

Alcohol Dehydrogenase↗
PubMed1991

In vivo footprinting reveals unique cis-elements and different modes of hypoxic induction in maize Adh1 and Adh2.

The transcriptional activation of maize alcohol dehydrogenase-1 (Adh1) and alcohol dehydrogenase-2 (Adh2) is accompanied by changes in the chromatin structure within the 5'-flanking region of each gene. The positions of DNA-binding factors bound to the 5'-flanking regions were determined by in vivo dimethyl sulfate footprinting of maize suspension cultures over 8 hours of hypoxic induction. In Adh1 there are two types of DNA-binding factors associated with the promoter region. One set of factors is constitutively associated with the cis-regulatory anaerobic response element, whereas two additional factors bind only after Adh1 has been induced by hypoxic stress. Returning hypoxically stressed cells to an aerobic environment restores the dimethyl sulfate footprint observed for the uninduced Adh1 gene. In contrast, all of the factors bound to the 5'-flanking region of Adh2 are constitutively present and unchanged by hypoxia. There is one footprint site common to both Adh1 and Adh2, but it is not an anaerobic response-like element.

Aerobiosis↗
PubMed1990

ARF-B(2): A Protein Complex that Specifically Binds to Part of the Anaerobic Response Element of Maize Adh 1.

Crude whole cell extracts from maize (Zea mays L.) suspension cells were examined for DNA binding proteins that specifically interact with a portion of the maize Adh 1 promoter that was previously shown to be in contact with a trans-acting factor in vivo. A 17 base pair, double-stranded oligonucleotide probe was constructed that centered around a strong in vivo dimethylsulfate footprint (B(2)) that coincides with part of the anaerobic response element (ARE). Gel retardation assays were used to characterize a major, specific DNA binding protein activity found in the crude extracts. The activity is present in both aerobic and hypoxically treated cultures and has been designated ARF-B(2) (ARE binding factor). ARF-B(2) appears to be a multicomponent complex, with a 54 kilodalton subunit termed ARF-B(2)alpha in primary contact with the target DNA.

Journal Article↗
PubMed1990

In vivo and in vitro characterization of protein interactions with the dyad G-box of the Arabidopsis Adh gene.

Expression of the alcohol dehydrogenase (Adh) and ribulose-1,5-bisphosphate carboxylase small subunit (RbcS) genes of higher plants is cell-type-specific and environmentally inducible. However, the tissues in which these two genes are expressed, their modes of induction, and their protein functions are quite distinct. Adh is expressed in non-green tissue, induced by anaerobiosis, and repressed in leaves. RbcS is only expressed in green tissue. An 8-base pair G-box element (5'-CCACGTGG-3') is associated with light-induced expression of RbcS and chalcone synthase. The same sequence is also present in the 5'-flanking region of Arabidopsis thaliana Adh, and this sequence is associated with a trans-acting factor in vivo. We report here that in vitro Adh G-box binding activity is present in crude whole cell extracts of both cell culture and leaves of Arabidopsis. The authenticity of in vitro Adh G-box binding is supported by in vivo and in vitro dimethylsulfate footprinting. A clear in vivo Adh G-box footprint occurs in cell cultures, but comparable in vivo binding to the Adh G-box does not occur in leaves. Therefore, there does not appear to be a direct correlation between the presence of the G-box factor in a tissue and its binding to the Adh G-box.

Alcohol Dehydrogenase↗