Biomedical subjects

J Chory

Publications and source records attributed to J Chory.

83 records · Page 5Linked to original sources

PubMed1990

Mutations in the DET1 gene affect cell-type-specific expression of light-regulated genes and chloroplast development in Arabidopsis.

When grown in the absence of light, the det1 mutant of Arabidopsis thaliana develops characteristics of a light-grown plant by morphological, cellular, and molecular criteria. Here, we show that recessive mutations at the DET1 locus also result in cell-type inappropriate accumulation of RNAs for light-regulated nuclear and chloroplast genes. det1 root plastids are differentiated into chloroplasts and are present in very high numbers in root cortex cells in contrast to the few starch-containing amyloplasts normally found in Arabidopsis roots. To assay the effect of the det1 mutation on the expression of photoregulated promoters, we used fusion constructs to stably transform wild-type and det1 mutants. We show that the three red-light-regulated chlorophyll a/b binding protein promoters are inappropriately expressed in the roots of det1 seedlings and the blue-light-controlled anthocyanin biosynthetic gene, chalcone synthase, is expressed ectopically in leaf mesophyll cells. These results, together with out previous findings, suggest that the DET1 gene product is a negatively acting regulatory molecule that is used in common by the light stimulus transduction pathway and by temporal or spatial regulatory signals in plants.

Blotting, Northern↗

PubMed1990

Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis.

Phenylalanine ammonia-lyase (PAL) is encoded by a small family of genes in Arabidopsis. We cloned and partially characterized one of these genes, PAL1. The deduced amino acid sequence is highly similar to PAL from bean, parsley, and rice. The promoter contains sequence elements homologous to two putative regulatory elements conserved among several phenylpropanoid genes. The regulation of the PAL1 gene was examined by analysis of beta-glucuronidase (GUS) activity in transgenic Arabidopsis containing PAL1-GUS gene fusions. The PAL1 promoter was activated early in seedling development and in adult plants was strongly expressed in the vascular tissues of roots and leaves, but was not active in the root tip or the shoot apical meristem. In flowers, expression was observed in sepals, anthers, and carpels, but not in petals. Transcripts encoded by the endogenous PAL genes and GUS transcripts from the PAL1-GUS gene fusion were induced by wounding, HgCl2-stress, and light. Analysis of the regulatory properties of 5' deleted promoters showed that the proximal region of the promoter to -290 was sufficient to establish the full tissue-specific pattern of expression and that the proximal region to -540 was responsive to environmental stimuli. Negative and positive elements were located between -1816 and -823 and between -823 and -290, respectively.

Amino Acid Sequence↗

PubMed1989

Arabidopsis thaliana mutant that develops as a light-grown plant in the absence of light.

The signal transduction pathways that lead to chloroplast biogenesis in plants are largely unknown. We describe here the identification and initial characterization of a novel genetic locus which fits the criteria of a regulatory gene located in a central pathway controlling light-mediated development. In the absence of light, these Arabidopsis thaliana mutants, designated det1 (de-etiolated 1), constitutively display many characteristics that are light-dependent in wild-type plants, including leaf and chloroplast development, anthocyanin accumulation, and accumulation of mRNAs for several light-regulated nuclear and chloroplast genes. The switch between dark and light growth modes thus appears to be a programmed step in a developmental pathway that is defined by det1. We suggest a model where the primary role of light on gene expression is mediated by the activation of leaf development. Further, the recessive nature of the det1 mutation implies that there is negative growth control on leaf development in dicotyledonous plants in the absence of light.

Anthocyanins↗

PubMed1987

Gibberellin-Induced Changes in the Populations of Translatable mRNAs and Accumulated Polypeptides in Dwarfs of Maize and Pea.

Two-dimensional gel electrophoresis was used to characterize the molecular mechanism of gibberellin-induced stem elongation in maize and pea. Dwarf mutants of maize (d-5) and pea (Progress No. 9) lack endogenous gibberellin (GA(1)) but become phenotypically normal with exogenous applications of this hormone. Sections from either etiolated maize or green pea seedlings were incubated in the presence of [(35)S] methionine for 3 hours with or without gibberellin. Labeled proteins from soluble and particulate fractions were analyzed by two-dimensional gel electrophoresis and specific changes in the patterns of protein synthesis were observed upon treatment with gibberellin. Polyadenylated mRNAs from etiolated or green maize shoots and green pea epicotyls treated or not with gibberellin (a 0.5 to 16 hour time course) were assayed by translation in a rabbit reticulocyte extract and separation of products by two-dimensional gel electrophoresis. Both increases and decreases in the levels of specific polypeptides were seen for pea and corn, and these changes were observed within 30 minutes of treatment with gibberellin. Together, these data indicate that gibberellin induces changes in the expression of a subset of gene products within elongating dwarfs. This may be due to changes in transcription rate, mRNA stability, or increased efficiency of translation of certain mRNAs.

Journal Article↗

PubMed1986

In vitro biosynthesis and membrane association of photosynthetic reaction center subunits from Rhodopseudomonas sphaeroides.

The reaction center of Rhodopseudomonas sphaeroides is an integral membrane protein complex responsible for primary photochemical charge separation in photosynthesis. We report the synthesis of two of the three subunits of the photosynthetic reaction center using a DNA-directed in vitro transcription-translation system prepared from R. sphaeroides. The in vitro-synthesized polypeptides, as resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, had apparent Mrs of 24,000 and 21,000 and were shown to be synthesized in equimolar amounts. This corresponds precisely to the in vivo reaction center subunits M and L, respectively. The in vitro-synthesized polypeptides were immunoprecipitated with antibody prepared against whole native reaction centers. In addition, the identity of the in vitro-synthesized polypeptides as L and M was verified by comparing the protease digestion products of in vivo- with in vitro-synthesized reaction center subunits. Both of the in vitro-synthesized polypeptides were also found to partition with the particulate material in the transcription-translation system and to associate with added membranes.

Bacterial Proteins↗

PubMed1985

DNA-directed in vitro synthesis and assembly of the form II D-ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides.

A biochemical analysis of the in vitro assembly of the form II ribulose-1,5-bisphosphate carboxylase/oxygenase from Rhodopseudomonas sphaeroides after transcription and translation from cloned DNA is presented. The predominant enzymatically active oligomeric forms of the in vitro-synthesized and -assembled ribulose-1,5-bisphosphate carboxylase are tetramers and hexamers. Assembly of the monomeric subunits to form active enzyme appears to be dependent on the presence of a minimum number of subunits in the cell extract. Assembly of ribulose-1,5-bisphosphate carboxylase also was observed when the protein-synthesizing extracts were prepared from cells which were partially derepressed for ribulose-1,5-bisphosphate carboxylase expression.

DNA, Bacterial↗

PubMed1985

Cloning and characterization of the gene product of the form II ribulose-1,5-bisphosphate carboxylase gene of Rhodopseudomonas sphaeroides.

We report the cloning and characterization of the gene product of the gene for the form II ribulose bisphosphate carboxylase from Rhodopseudomonas sphaeroides. We present evidence that the form II enzyme is encoded by a single gene in R. sphaeroides; however, this gene does hybridize to a second chromosomal locus.

Base Sequence↗

PubMed1985

Structure and physical map of Rhodopseudomonas sphaeroides bacteriophage RS1 DNA.

We analyzed, by restriction endonuclease mapping and electron microscopy, the genome of the lytic Rhodopseudomonas sphaeroides-specific bacteriophage RS1 and characterized it as a linear molecule of approximately 60 to 65 kilobases. When the DNA from purified phage particles was examined by several independent methods, considerable size heterogeneity was apparent in the RS1 DNA. This size heterogeneity was concluded to be of biological origin, was independent of the specific host strain used to propagate virus, and was not due to the presence of host DNA within or nonspecifically associated with purified virions. In addition, treatment of RS1 DNA with either BAL 31 nuclease or DNA polymerase I Klenow fragment revealed that several distinct regions exist within the viral chromosome which contain free 3' hydroxyl groups. A restriction endonuclease map of the RS1 genome was constructed by using the restriction endonucleases EcoRI, ClaI, KpnI, BamHI, MluI, SmaI, and BclI; thereby allowing the positioning of some 40 restriction sites within the viral genome. The results are discussed in terms of the significance and the possible biological origin of the unique features discovered within the phage RS1 DNA.

Bacteriophages↗

PubMed1984

Induction of the photosynthetic membranes of Rhodopseudomonas sphaeroides: biochemical and morphological studies.

Cells of Rhodopseudomonas sphaeroides grown in a 25% O2 atmosphere were rapidly subjected to total anaerobiosis in the presence of light to study the progression of events associated with the de novo synthesis of the inducible intracytoplasmic membrane (ICM). This abrupt change in physiological conditions resulted in the immediate cessation of cell growth and whole cell protein, DNA, and phospholipid accumulation. Detectable cell growth and whole cell protein accumulation resumed ca. 12 h later. Bulk phospholipid accumulation paralleled cell growth, but the synthesis of individual phospholipid species during the adaptation period suggested the existence of a specific regulatory site in phospholipid synthesis at the level of the phosphatidylethanolamine methyltransferase system. Freeze-fracture electron microscopy showed that aerobic cells contain small indentations within the cell membrane that appear to be converted into discrete ICM invaginations within 1 h after the imposition of anaerobiosis. Microscopic examination also revealed a series of morphological changes in ICM structure and organization during the lag period before the initiation of photosynthetic growth. Bacteriochlorophyll synthesis and the formation of the two light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides (B800-850 and B875) occurred coordinately within 2 h after the shift to anaerobic conditions. Using antibodies prepared against various ICM-specific polypeptides, the synthesis of reaction center proteins and the polypeptides associated with the B800-850 complex was monitored. The reaction center H polypeptide was immunochemically detected at low levels in the cell membrane of aerobic cells, which contained no detectable ICM or bacteriochlorophyll. The results are discussed in terms of the oxygen-dependent regulation of gene expression in R. sphaeroides and the possible role of the reaction center H polypeptide and the cell membrane indentations in the site-specific assembly of ICM pigment-protein complexes during the de novo synthesis of the ICM.

Aerobiosis↗

PubMed1983

Light-dependent regulation of the synthesis of soluble and intracytoplasmic membrane proteins of Rhodopseudomonas sphaeroides.

Cells of Rhodopseudomonas sphaeroides grown under saturating light conditions (30 W/m2) and then shifted to low light intensity (3 W/m2) required 2.5 h to adapt to the new lower light conditions. After the shift, cell growth, whole cell protein accumulation, and bacteriochlorophyll accumulation ceased immediately. Approximately midway into the adaptation period, bacteriochlorophyll synthesis commenced at a new, higher rate, which continued through the beginning of the low-light growth period until new steady-state levels were reached. Immediately after the downshift, the rate of cellular protein synthesis declined to 22% of its preshift rate. Pulse-labeling of protein throughout the adaptation period and comparison with a steady-state prelabel culture revealed that synthesis of two of the three light-harvesting proteins, as well as two additional high-molecular-weight photosynthetic membrane proteins, was derepressed three- to fivefold compared with bulk cellular protein. Finally, the synthesis of at least three soluble proteins showed light-dependent regulation after the light downshift. These results are discussed in terms of the light-dependent regulation of synthesis of the photosynthetic membrane macromolecular components and the division of protein synthesis between the photosynthetic membranes and the soluble cell phase.

Bacterial Proteins↗

PubMed1982

The in vitro transcription-translation of DNA and RNA templates by extracts of Rhodopseudomonas sphaeroides. Optimization and comparison of template specificity with Escherichia coli extracts and in vivo synthesis.

A DNA-directed coupled transcription-translation system has been developed in cell-free extracts from the facultative phototroph, Rhodopseudomonas sphaeroides. The in vitro protein-synthesizing system was active when prepared from either chemoheterotrophically or photoheterotrophically grown cells. Optimal activity was dependent upon: use of extracts prepared freshly from early exponential phase cells, the method of cell breakage, and the length of preincubation of the extract (S-30), as well as the concentrations of S-30, template, and cations. The R. sphaeroides cell-free system was compared to one prepared from Escherichia coli. DNA templates tested included R. sphaeroides phage RS1 DNA and E. coli phages T4 and T7 DNA, as well as plasmids RSF1010, pBR322, pSL25 (a pBR322 derivative), and a chimeric plasmid of pSL25 and RSF1010. One RNA template, phage R17, was also employed to test translational fidelity. Transcriptional-translational specificity was observed between R. sphaeroides and E. coli and these observations are discussed in terms of differential gene expression among phylogenetically distinct groups of bacteria.

DNA, Bacterial↗