Search PubMed⌕ Search

Biomedical subjects

R J Ferl

Publications and source records attributed to R J Ferl.

At least 37 records · Page 2Linked to original sources
PubMed1997

The Arabidopsis 14-3-3 multigene family.

The 14-3-3 proteins are ubiquitous eukaryotic proteins and are encoded by a gene family in many species. We examined the 14-3-3 gene family in Arabidopsis thaliana and found that it contains 10 members. Four new cDNAs, GF14 epsilon, GF14 kappa, GF14 mu, and GF14 nu, and two new genomic clones of GF14 phi and GF14 nu were isolated and characterized. Together with the six previously described 14-3-3 isoforms in Arabidopsis, they constitute a complete family of 10 distinct 14-3-3 proteins of 248 to 268 amino acids. Phylogenetic analysis revealed the presence of two ancient, distinct 14-3-3 gene classes in Arabidopsis and other plants. The epsilon forms diverged early from the other plant isoforms, and plant 14-3-3 genes displayed a different evolutionary course from that of mammals.

14-3-3 Proteins↗
PubMed1996

14-3-3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform-specific manner and reduce dephosphorylation of Ser-543 by endogenous protein phosphatases.

Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: omega> chi> upsilon>>> phi, psi.

14-3-3 Proteins↗
PubMed1996

Plant metabolism: enzyme regulation by 14-3-3 proteins.

14-3-3 proteins have been found to regulate the plant enzyme nitrate reductase by reversible phosphoserine binding. Plant plasma-membrane H(+)-ATPases, transporters that are activated by the phytotoxin fusicoccin, appear to be regulated in a similar fashion.

14-3-3 Proteins↗
PubMed1996

Four Arabidopsis thaliana 14-3-3 protein isoforms can complement the lethal yeast bmh1 bmh2 double disruption.

The 14-3-3 proteins comprise a family of highly conserved proteins with multiple functions, most of which are related to signal transduction. Four isoforms from the plant Arabidopsis thaliana were able to complement the lethal disruption of the two Saccharomyces cerevisiae genes encoding 14-3-3 proteins; one complemented very poorly and one did not complement. However, the expression of the latter two isoforms was very low. These results show that at least four of the six A. thaliana isoforms are able to perform the same function(s) as the yeast 14-3-3 proteins.

14-3-3 Proteins↗
PubMed1996

Transcription factor veracity: is GBF3 responsible for ABA-regulated expression of Arabidopsis Adh?

Assignment of particular transcription factors to specific roles in promoter elements can be problematic, especially in systems such as the G-box, where multiple factors of overlapping specificity exist. In the Arabidopsis alcohol dehydrogenase (Adh) promoter, the G-box regulates expression in response to cold and dehydration, presumably through the action of abscisic acid (ABA), and is bound by a nuclear protein complex in vivo during expression in cell cultures. In this report, we test the conventional wisdom of biochemical approaches used to identify DNA binding proteins and assess their specific interactions by using the G-box and a nearby half G-box element of the Arabidopsis Adh promoter as a model system. Typical in vitro assays demonstrated specific interaction of G-box factor 3 (GBF3) with both the G-box and the half G-box element. Dimethyl sulfate footprint analysis confirmed that the in vitro binding signature of GBF3 essentially matches the footprint signature detected in vivo at the G-box. Because RNA gel blot data indicated that GBF3 is itself induced by ABA, we might have concluded that GBF3 is indeed the GBF responsible in cell cultures for binding to the Adh G-box and is therefore responsible for ABA-regulated expression of Adh. Potential limitations of this conclusion are exposed by the fact that other GBFs bind the G-box with the same signature as GBF3, and subtle differences between in vivo and in vitro footprint signatures indicate that factors other than or in addition to GBF3 interact with the half G-box element.

Abscisic Acid↗
PubMed1996

Molecular organization and tissue-specific expression of an Arabidopsis 14-3-3 gene.

The 14-3-3 proteins, originally described as mammalian brain proteins, are ubiquitous in eukaryotes. We isolated an Arabidopsis 14-3-3 gene, designated GRF1-GF14 chi (for general regulatory factor1-G-box factor 14-3-3 homolog isoform chi), and characterized its expression within plant tissues. Sequence comparison of the GRF1-GF14 chi genomic clone with other 14-3-3 proteins demonstrated that the extreme conservation of 14-3-3 residues in several domains is encoded by the first three exons. The highly variable C-terminal domain is encoded by a divergent fourth exon that is unique among 14-3-3 homologs, suggesting that exon shuffling might confer gene-specific functions among the isoforms. The anatomical distribution and developmental expression of the Arabidopsis 14-3-3 protein were examined in transgenic plants carrying a GRF1-GF14 chi promoter-beta-glucuronidase construct. GF14 chi promoter activity was observed in the roots of both seedlings and mature plants. In immature flowers, GF14 chi promoter activity was localized to the buds. However, as the flowers matured, GF14 chi promoter activity was restricted to the stigma, anthers, and pollen. In immature siliques, GF14 chi promoter activity was initially localized to styles and abscission zones but was subsequently observed throughout mature siliques. In situ hybridization demonstrated that GF14 chi mRNA expression was prominent in epidermal tissue of roots, petals, and sepals of flower buds, papillae cells of flowers, siliques, and endosperm of immature seeds. Thus, plant 14-3-3 gene expression exhibits cell- and tissue-specific localization rivaling that observed for 14-3-3 proteins within the mammalian brain.

14-3-3 Proteins↗
PubMed1995

Two cDNA clones encoding 14-3-3 homologs from tomato fruit.

Two tomato cDNA clones with homology to the 14-3-3 family of proteins were identified through immunoscreening a ripening tomato fruit library (Clontech). These two clones share approx. 71% identity at the nucleotide level and 84% identity at the deduced amino acid level, with radical amino acid substitutions clustering at the acidic carboxy-terminus. Southern hybridization data indicate that each clone represents a unique gene. Distinct transcript accumulation patterns during tomato fruit ripening together with the homology to brain regulatory proteins suggest potential involvement in fruit development.

14-3-3 Proteins↗
PubMed1995

Trans-Pacific migrations of the loggerhead turtle (Caretta caretta) demonstrated with mitochondrial DNA markers.

Juvenile loggerhead turtles (Caretta caretta) have recently been documented in the vicinity of Baja California and thousands of these animals have been captured in oceanic fisheries of the North Pacific. The presence of loggerhead turtles in the central and eastern North Pacific is a prominent enigma in marine turtle distribution because the nearest documented nesting concentrations for this species are in Australia and Japan, over 10,000 km from Baja California. To determine the origin of the Baja California feeding aggregate and North Pacific fishery mortalities, samples from nesting areas and pelagic feeding aggregates were compared with genetic markers derived from mtDNA control region sequences. Overall, 57 of 60 pelagic samples (95%) match haplotypes seen only in Japanese nesting areas, implicating Japan as the primary source of turtles in the North Pacific Current and around Baja California. Australian nesting colonies may contribute the remaining 5% of these pelagic feeding aggregates. Juvenile loggerhead turtles apparently traverse the entire Pacific Ocean, approximately one-third of the planet, in the course of developmental migrations, but mortality in high-seas fisheries raises concern over the future of this migratory population.

Animals↗
PubMed1995

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.

Alcohol Dehydrogenase↗
PubMed1995

The Arabidopsis Adh gene exhibits diverse nucleosome arrangements within a small DNase I-sensitive domain.

The alcohol dehydrogenase (Adh) gene from Arabidopsis shows enhanced sensitivity to DNase I in cells that express the gene. This generalized sensitivity to DNase I is demarcated by position -500 on the 5' side and the end of the mRNA on the 3' side. Thus, the gene defined as the promoter and mRNA coding region corresponds very closely in size with the gene defined as a nuclease-sensitive domain. This is a remarkably close correspondence between a sensitive domain and a eukaryotic transcriptional unit, because previously reported DNase I-sensitive domains include large regions of DNA that are not transcribed. Nucleosomes are present in the coding region of the Adh gene when it is expressed, indicating that the transcriptional elongation process causes nucleosome disruption rather than release of nucleosomes from the coding region. In addition, the regulatory region contains a loosely positioned nucleosome that is separated from adjacent nucleosomes by internucleosomic DNA segments longer than the average linker DNA in bulk chromatin. This specific array of nucleosomes coexists with bound transcription factors that could contribute to the organization of the nucleosome arrangement. These results enhance our understanding of the complex interactions among DNA, nucleosomes, and transcription factors during gene expression in plants.

Alcohol Dehydrogenase↗
PubMed1994

Expression of active, processed ricin in transgenic tobacco.

The cDNA encoding the plant toxin precursor preproricin was introduced into tobacco via Agrobacterium tumefaciens-mediated gene transfer. Transgenic plants were assayed for type II ribosome-inactivating protein expression and activity. Western blot analysis of soluble leaf extracts using anti-ricin a-chain (RTA) antibodies identified 34- and 32-kDa proteins, which were electrophoretically indistinguishable from castor seed RTA. Analysis with anti-ricin b-chain (RTB) antibodies identified both a 34-kDa protein major band, which co-migrated with castor seed RTB, and a 30-kDa protein minor band. Enzyme-linked immunoassay of the transgenic leaf extracts with anti-RTA and anti-RTB indicated microgram per gram production on a fresh weight basis of soluble extractable recombinant ricin. Sugar binding enzyme-linked immunoassay employing an immobilized glycoprotein, asialofetuin, and anti-RTB antibodies confirmed the characteristic type II ribosome-inactivating protein galactose binding lectin activity of the recombinant ricin. The enzymatic activity of recombinant ricin was characterized for cell-free translation inhibition, as well as for overall cytotoxicity. A 50% inhibitory dose of 3 x 10(-11) M was observed for the immunoreactive leaf extract material using a rabbit reticulocyte translation inhibition assay, while a 50% lethal dose of 1 x 10(-12) M was calculated with human T-lymphotropic virus-1 infected leukemic T-cells.

Agrobacterium tumefaciens↗
PubMed1994

A single Arabidopsis GF14 isoform possesses biochemical characteristics of diverse 14-3-3 homologues.

Arabidopsis cDNA clones of GF14 proteins originally were isolated on the basis of their association with the G-box DNA/protein complex by a monoclonal antibody screening approach. GF14 proteins are homologous to the 14-3-3 family of mammalian proteins. Here we demonstrate that recombinant GF14 omega, one member of the Arabidopsis GF14 protein family, is a dimeric protein that possesses many of the attributes of diverse mammalian 14-3-3 homologues. GF14 omega activates rat brain tryptophan hydroxylase and protein kinase C in a manner similar to the bovine 14-3-3 protein. It also activates exoenzyme S of Pseudomonas aeruginosa as does bovine brain factor activating exoenzyme S (FAS), which is itself a member of 14-3-3 proteins. In addition, GF14 omega binds calcium, as does the human 14-3-3 homologue reported to be a phospholipase A2. These results indicate that a single isoform of this plant protein family can have multiple functions and that individual GF14 isoforms may have multiple roles in mediating signal transductions in plants. However, GF14 omega does not regulate growth in an in vivo test for functional similarity to the yeast 14-3-3 homologue, BMH1. Thus, while a single plant GF14 isoform can exhibit many of the biochemical attributes of diverse mammalian 14-3-3 homologues, open questions remain regarding the physiological functions of GF14/14-3-3 proteins.

14-3-3 Proteins↗
PubMed1994

Partial sequencing and mapping of clones from two maize cDNA libraries.

As one component of a maize genome project, we report the analysis of a number of randomly selected cDNAs, by a combination of measuring mRNA expression, 'single-pass' sequencing (SPS), and genome mapping. Etiolated seedling (490) and membrane-free polysomal endosperm cDNA clones (576) were evaluated for their transcription levels by hybridizing with a probe prepared from total mRNA and categorized as corresponding to abundantly or rarely expressed mRNAs and as either constitutive or tissue-specific. A total 313 clones from the two libraries were submitted to 'single-pass' sequencing from the presumed 5' end of the mRNA and the nucleotide sequence compared with the GenBank database. About 61% of the clones showed no significant similarities within GenBank, 14% of the clones exhibited a high degree of similarity, while the remaining 25% exhibited a lesser degree of similarity. The chromosomal location of more than 300 clones was determined by RFLP mapping using standard populations. The results demonstrate that a combination of analyses provides synergistic information in eventually deducing the actual function of these types of clones.

Chromosomes↗
PubMed1994

Evolutionary implications of the family of 14-3-3 brain protein homologs in Arabidopsis thaliana.

The GF14 family of proteins in Arabidopsis thaliana consists of a homologous group of polypeptides ranging in size from approximately 27 kDa to approximately 32 kDa. As a group, GF14 proteins are also homologous to a family of mammalian proteins most commonly referred to as 14-3-3 proteins. Several distinct and different biochemical activities have been historically attributed to the various isoforms of the mammalian 14-3-3 proteins. These data present the possibility that the various activities are performed by functionally distinct lineages of the gene family. Here we present phylogenetic analyses based on the derived amino acid sequences of five GF14 isoforms expressed in Arabidopsis suspension-cultured cells. A high degree of sequence integrity is apparent in the various Arabidopsis isoforms, and the overall structures of the plant forms are quite conserved with regard to the structures of the known mammalian forms. These gene phylogenies indicate no evolutionary conservation of specific isoform lineages within both plants and animals. Rather, the evolutionary history of this protein appears to be characterized by a separate radiation of plant and animal forms from a common ancestral sequence. Even though the plant and animal forms have evolved independently since that ancestral split, large domains are conserved in both major lineages.

14-3-3 Proteins↗
PubMed1994

Transcriptional regulation of environmentally inducible genes in plants by an evolutionary conserved family of G-box binding factors.

1. In reviewing a number of the most intensely studied environmentally inducible promoters it becomes clear that the presence of two cis-acting elements are critical for promoter activity, one of which is the G-box (CCACGTGG). A mutation in one of the two elements abolishes or severely reduces the ability of the promoter to respond to environmental changes. The sequence of the second cis-acting element, positioned nearby the G-box, is not conserved among the different inducible promoters, but may be similar among promoters induced by the same signal. The spacing between the G-box and the second cis-acting element appears to be critical, suggesting a direct interaction between the respective binding factors. We speculate on a potential role of the G-box promoter element in the signal induction of promoter activity. 2. From a number of plant species nuclear proteins interacting with the G-box have been identified. Recently, G-box Binding Factors (GBF) have been isolated by screening cDNA expression libraries with a characterized G-box cis-acting element as DNA probe. The deduced amino acid sequence of the GBF clones revealed that they possess the features of the basic leucine zipper class of trans-acting factors. By amino acid sequence comparison and limited mutational analysis, we define amino acids critical for G-box binding specificity. All GBFs isolated to date have a conserved proline-rich domain involved in transcriptional activation. A number of GBFs are inducible by a particular environmental signal. 3. Recently, a protein designated GF14 has been isolated that is associated with the GBF protein complex. The protein has homology to mammalian brain specific proteins, which seem to function as regulators of phosphorylation events. GBF activity is regulated by phosphorylation. The GF14 proteins may therefore impose an additional control on gene expression.

Amino Acid Sequence↗
PubMed1994

Analysis of expressed sequence tags from Plasmodium falciparum.

An initiative was undertaken to sequence all genes of the human malaria parasite Plasmodium falciparum in an effort to gain a better understanding at the molecular level of the parasite that inflicts much suffering in the developing world. 550 random complimentary DNA clones were partially sequenced from the intraerythrocytic form of the parasite as one of the approaches to analyze the transcribed sequences of its genome. The sequences, after editing, generated 389 expressed sequence tag sites and over 105 kb of DNA sequences. About 32% of these clones showed significant homology with other genes in the database. These clones represent 340 new Plasmodium falciparum expressed sequence tags.

Animals↗