Biomedical subjects

R J Ferl

Publications and source records attributed to R J Ferl.

67 records · Page 4Linked to original sources

PubMed1990

Characterization of the Arabidopsis Adh G-box binding factor.

Protein-DNA interaction at an inverted repeat of the sequence 5'-GTGG-3' (G-box) has been associated with the transcription of several plant genes [Giuliano, G., et al. (1988). Proc. Natl. Acad. Sci. USA 85, 7089-7093; Ferl, R.J., and Laughner, B.H. (1989). Plant Mol. Biol. 12, 357-366; Schulze-Lefert, P., et al. (1989). EMBO J. 8, 651-656]. We characterized the binding of the Arabidopsis G-box binding factor (GBF) from whole-cell extracts and fractionated extracts to the G-box of alcohol dehydrogenase (Adh) using gel mobility shift assays. DNase I footprinting localized the region of GBF/G-box interaction to two sites, one apparent high-affinity binding site (-227 to -201) and a possible low-affinity binding site (-193 to -182). DNA-protein cross-linking demonstrated that the G-box is bound by proteins of two sizes, 31 kilodaltons and 18 kilodaltons. In addition, we found that in vitro the interaction of GBF from Arabidopsis suspension cultures or leaves with the Adh G-box is indistinguishable, and that there is evidence of multiple protein-protein interactions.

Alcohol Dehydrogenase↗

PubMed1989

Increased gene expression by the first intron of maize shrunken-1 locus in grass species.

The first intron of the shrunken-1 (Sh1) locus of maize was incorporated into constructs containing the chloramphenicol acetyltransferase gene (CAT) coupled with the nopaline synthase 3' polyadenylation signal. Transcription was driven with the 35S promoter of the cauliflower mosaic virus (CaMV) or the Sh1 promoter of maize. Transient gene expression was monitored following electroporation into protoplasts of Panicum maximum (guineagrass), Pennisetum purpureum (napiergrass), or Zea mays (maize). The 1028 base pair intron increased gene expression in cells of each species when transcription was driven with the 35S promoter. Eleven to 91-fold increases were observed. Expression levels observed in maize were two and eight times those observed in napiergrass and guineagrass, respectively. The 35S promoter gave CAT activity 10 to 100 times that observed with the Sh1 promoter. Whereas expression driven by the 35S promoter was reproducible, that observed with the Sh1 promoter proved quite variable. In similar constructs the first intron of the alcohol dehydrogenase-1 (Adh1) gene of maize led to increased gene expression of only 7 to 10% of that observed with the Sh1 first intron. The increased level of gene expression caused by the Sh1 first intron is approximately 10 times higher than that caused by any other plant introns that have been used. Thus, the Sh1 first intron may prove quite useful in increasing expression of foreign genes in monocots and possibly other plants.

Journal Article↗

PubMed1988

Nucleotide sequence of an actin gene from Arabidopsis thaliana.

An 1830-bp genomic DNA segment containing an Arabidopsis thaliana actin gene, AAc1, has been cloned and sequenced. The AAc1 gene is present as a single-copy gene, but at least two other actin-like genes have been detected. Comparison of the nucleotide sequence of AAc1 with other cloned plant actin genes reveals four exons separated by three introns conservatively located in all plant actin genes. The deduced amino acid sequence has also been compared with actin protein sequences from other plants.

Actins↗

PubMed1988

The complete nucleotide sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila: phylogenetic implications.

The DNA sequence of the small-subunit ribosomal RNA coding region for the cycad Zamia pumila L. was determined. The Zamia small-subunit rRNA was found to be 1813 nucleotides in length and approximately 92% identical to published angiosperm small-subunit rRNA sequences. Conserved regions interspersed with variable regions are observed corresponding to those found in other eukaryotic small-subunit sequences. Using representatives from protist, fungal, plant, and animal groups, a distance matrix was constructed of average nucleotide substitution rates for pairs of organisms. Phylogenetic trees were inferred from similarities between sequences. The sequence of Zamia represents the earliest divergence from the higher plant lineage reported to date for small-subunit rRNA data. Inferred phylogenies also support a monophyletic origin for the angiosperms consistent with studies citing phenotypic characters.

Base Sequence↗

PubMed1987

In vivo detection of regulatory factor binding sites in the 5' flanking region of maize Adh1.

In vivo dimethyl sulfate footprinting experiments have been used to locate the binding sites of regulatory molecules in the TATA proximal portion of the maize alcohol dehydrogenase-1 gene. In several tissues and organs, Adh1 is transcriptionally induced by anaerobic stress, and the various alleles of Adh1 can show a quantitative differential response to induction. Two regulatory molecules were found to be bound to the promoter only when the gene was induced. The binding sites for these factors mapped to regions located at -100 to -108 and -186 to -190 relative to the site of transcript initiation. Two additional regulatory molecules were bound to sites located at positions -117 to -120 and -138 to -145 regardless of the state of transcription. However, the factor bound at the -138 to -145 region was found to alter its binding characteristics when the gene was induced.

Alcohol Dehydrogenase↗

PubMed1987

Constitutive and anaerobically induced DNase-I-hypersensitive sites in the 5' region of the maize Adh1 gene.

DNase-I-hypersensitive sites have been characterized in a plant gene, maize Adh1 (which encodes alcohol dehydrogenase 1). It has been generally recognized in animal genes that the chromatin of the 5' flanking region can be characterized by the accessibility of its DNA to the nuclease DNase I (EC 3.1.21.1), indicating which areas in the promoter are "open" to nuclear factors. The 5' region of the maize Adh1 gene contains two distinct DNase-I-hypersensitive regions, one constitutively present from position -160 to -700 and one that is anaerobically induced from position -35 to -150. The constitutive region contains three major hypersensitive sites, one of which corresponds in part to a region of potential Z-DNA. The induced hypersensitive region includes TATAA at -38 and CAAT at -100 as well as other potential regulatory sequences.

Journal Article↗

PubMed1986

Somatic hybridization in the Gramineae: Saccharum officinarum L. (sugarcane) and Pennisetum americanum (L.) K. Schum. (pearl millet).

Somatic hybrid cell lines with embryogenic capacity were obtained by fusion of protoplasts isolated from an embryogenic cell line of sugarcane with inactivated protoplasts of an S-(2-aminoethyl)-L-cysteine (Aet-Cys)-resistant cell line of pearl millet. Initial selection for putative hybrids was performed by plating protoplast-derived microcolonies onto medium containing 300 mug of Aet-Cys per ml. Calli growing in the presence of Aet-Cys were screened with respect to their electrophoretic pattern of phosphogluconate dehydrogenase (6-phospho-D-gluconate; EC 1.1.1.44), a dimeric enzyme. The somatic hybrid calli showed both parental homodimer bands plus an intermediate heterodimer band. Somatic hybrid lines were characterized further by electrophoretic patterns of shikimate dehydrogenase (shikimate; EC 1.1.1.25) and arylesterase (aryl-ester hydrolase; EC 3.1.1.2). All cell lines expressed the nuclear genomes of both parents. Use of a maize ribosomal DNA probe allowed the identification of diagnostic ribosomal DNA fragments in Xba I digests of total DNA in both parents and the somatic hybrids. The somatic hybrid lines carried both sugarcane and pearl millet ribosomal DNA fragments. The somatic hybrid calli formed somatic embryos. The banding pattern of phosphogluconate dehydrogenase from the somatic embryos was similar to that of the somatic hybrid calli.

Journal Article↗

PubMed1985

Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.

Alcohol Dehydrogenase↗

PubMed1985

Ultrastructural correlates of anaerobic stress in corn roots.

Cytological events in Zea mays root meristem were followed during 26 hr of anaerobic treatment. From 8 to 14 hr, mitochondria swelled drastically, Golgi apparatus actively produced vesicles, endoplasmic reticulum proliferated, and chromatin strongly condensed. Plastids appeared normal. By 26 hr, however, Golgi activity receded, mitochondria assumed long, polymorphic shapes, chromatin partly dispersed, and plastids swelled. ER remained prominent, and the cytoplasm contained long fibrous inclusions. This preliminary study emphasizes the need to examine quantitatively all cellular organelles periodically for longer periods when following events of stress or pathology. Our observations corroborate scattered reports in the literature for single organelles under anaerobic stress and represent the first set of correlated observations on the entire spectrum of cellular events.

Anaerobiosis↗

PubMed1984

Molecular analysis of the alcohol dehydrogenase (Adh1) gene of maize.

A cDNA clone of maize Adh1 which contains the entire protein coding region of the gene has been constructed. The protein sequence predicted from the nucleotide sequence is in agreement with limited protein sequencing data for the ADH1 enzyme. An 11.5 kb genomic fragment containing the Adh1 gene has been isolated using the cDNA clone as a probe, and the gene region fully sequenced. The gene is interrupted by 9 introns, their junction sequences fitting the animal gene consensus sequence. Within the gene there is a triplication of a segment (104 bp) spanning an intron-exon junction. Presumptive promoter elements have been identified and are similar in nucleotide sequence and location, relative to the start of transcription, to those of other plant and animal genes. No recognizable poly(A+) addition signal is evident. Comparison of the nucleotide sequences of the cDNA (derived from an Adh1 -F allele) and genomic (derived from an Adh1 -S allele) clones has identified an amino acid difference consistent with the observed difference in electrophoretic mobility of the two enzymes. The maize ADH1 amino acid sequence is 50% homologous to that of horse liver ADH but is only 20% homologous to yeast ADH.

Alcohol Dehydrogenase↗

PubMed1982

cDNA cloning and induction of the alcohol dehydrogenase gene (Adh1) of maize.

cDNA clones of Adh1, one of two genes encoding alcohol dehydrogenase (ADH; alcohol:NAD(+) oxidoreductase, EC 1.1.1.1) in the maize genome, have been isolated. They were derived from mRNA extracted from anaerobically treated roots of maize seedlings. Identification was initially made on the basis of molecular weight and electrophoretic properties of the in vitro polypeptide obtained in hybridization-release-translation experiments. The identification was confirmed by antibody precipitation and by the use of maize stocks having different genetic constitutions at the Adh1 locus. The sequence of the longest cDNA segment, approximately 900 base pairs, was determined and appears to code for 168 COOH-terminal amino acids and to have a 3' nontranslated region of 364 base pairs. Reverse Southern hybridizations established that two different Adh1-S stocks produce a mRNA of 1,650 nucleotides, whereas an additional mRNA of 1,750 nucleotides is produced in three Adh1-F stocks. A 50-fold increase in Adh1 mRNA level occurs during anaerobiosis, reaching a maximum at 5 hr. Return to aerobic conditions indicates a half-life of more than 18 hr for the anaerobically induced Adh1 mRNA.

Journal Article↗

PubMed1980

In vitro translation of maize ADH: evidence for the anaerobic induction of mRNA.

Total cellular RNA from anaerobically stressed maize seedling roots was used to stimulate in vitro translation of authentic maize alcohol dehydrogenase (ADH) in a rabbit reticulocyte lysate system. Total products from such reactions were displayed on NEPHGE-SDS two-dimensional gels and the Adhl-specific translation products were identified by using RNA from sib seedlings segregating for Adhl charge and size variants. The application of a rapid RNA isolation procedure allowed the efficient isolation of biologically active RNAs from small amonts of seedling material. Maize ADHs translated in vitro are identical in size to in vivo ADH. Further, no ADH was detected in the products of an in vitro translation reaction stimulated by total RNA from aerobically grown seedlings. This suggests that induction of ADH protein by anaerobic stress is accomplished by production of Adh mRNA rather than activation of sequestered mRNA. The mRNAs for maize ADH1 and ADH2 are among a small class of mRNAs induced during anaerobiosis.

Alcohol Oxidoreductases↗

PubMedDate not supplied

Global phylogeography of the ridley sea turtles (Lepidochelys spp.) as inferred from mitochondrial DNA sequences.

The Kemp's ridley sea turtle (Lepidochelys kempi) is restricted to the warm temperate zone of the North Atlantic Ocean, whereas the olive ridley turtle (L. olivacea) is globally distributed in warm-temperate and tropical seas, including nesting colonies in the North Atlantic that nearly overlap the range of L. kempi. To explain this lopsided distribution, Pritchard (1969) proposed a scenario in which an ancestral taxon was divided into Atlantic and Pacific forms (L. kempi and L. olivacea, respectively) by the Central American land bridge. According to this model, the olive ridley subsequently occupied the Pacific and Indian Oceans and recently colonized the Atlantic Ocean via southern Africa. To assess this biogeographic model, a 470 bp sequence of the mtDNA control region was compared among 89 ridley turtles, including the sole L. kempi nesting population and 7 nesting locations across the range of L. olivacea. These data confirm a fundamental partition between L. olivacea and L. kempi (p = 0.052-0.069), shallow separations within L. olivacea (p = 0.002-0.031), and strong geographic partitioning of mtDNA lineages. The most divergent L. olivacea haplotype is observed in the Indo-West Pacific region, as are the central haplotypes in a parsimony network, implicating this region as the source of the most recent radiation of olive ridley lineage. The most common olive ridley haplotype in Atlantic samples is distinguished from an Indo-West Pacific haplotype by a single nucleotide substitution, and East Pacific samples are distinguished from the same haplotype by two nucleotide substitutions. These shallow separations are consistent with the recent invasion of the Atlantic postulated by Pritchard (1969), and indicate that the East Pacific nesting colonies were also recently colonized from the Indo-West Pacific region. Molecular clock estimates place these invasions within the last 300,000 years.

Animals↗