Search PubMed⌕ Search

Biomedical subjects

L Bogorad

Publications and source records attributed to L Bogorad.

At least 55 records · Page 3Linked to original sources
PubMed1989

Visualization of antibody binding to the photosynthetic membrane: the transmembrane orientation of cytochrome b-559.

We have used immuno-gold labeling and electron microscopy to study the topography of thylakoid membrane polypeptides. Thylakoid vesicles formed by passage through a French press were adsorbed onto a plastic film supported by an electron microscope grid and processed for single or double immuno-gold labeling. After shadowing with platinum, the inside-out and right-side-out vesicles were identified by their distinctive morphologies. Right-side-out vesicles were labeled by a monoclonal antibody recognizing an epitope located in the trypsin-cleaved, N-terminal portion of the LHC II apoprotein, and by an antibody to CF1. A monoclonal antibody to the alpha-subunit of cytochrome b-559 reacted with a synthetic tridecapeptide corresponding to the C-terminal portion of the polypeptide. Both this antibody and a polyclonal antibody to the synthetic peptide labeled inside-out vesicles exclusively, indicating that the polypeptide C-terminus was exposed on the lumenal (exoplasmic) surface of the membrane.

Amino Acid Sequence↗
PubMed1989

Characterization of the ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803.

The ndhC and ORF159 genes of the maize plastid DNA (ptDNA) were sequenced and maize ORF159 was used to screen a library of genomic DNA of the blue-green alga Synechocystis sp. PCC 6803. The cyanobacterial gene homologous to ORF159 (ORF157) was isolated and sequenced. In sequencing the region upstream of ORF157, reading frames with homology to the ndhC and psbG genes of maize ptDNA were identified. The ndhC and psbG genes overlap in the ptDNAs of maize, tobacco and Marchantia polymorpha, but are separated by a noncoding spacer in Synechocystis. Northern blot analysis showed that the ndhC, psbG and ORF157/159 genes are cotranscribed in maize and Synechocystis. The three genes occur in the same order in ptDNA of maize, tobacco, and M. polymorpha as in Synechocystis 6803. The amino acid sequences of the NDH-C, PSII-G and the ORF157/159 proteins deduced from the maize genes are 65%, 52% and 53% homologous to those of Synechocystis. However, the cyanobacterial and higher plant NDH-C protein sequences are only 23% homologous to the mitochondrial NDH-3 protein. Protein products of in vitro transcription/translation of the Synechocystis transcription unit had apparent molecular masses of 6 kDa (NDH-C), 25 kDa (PSII-G) and 22 kDa (ORF157) on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis. If these are components of an NADH dehydrogenase, cyanobacteria appear to resemble mitochondria more than they do Escherichia coli and Rhodopseudomonas capsulata with regard to this enzyme complex.

Amino Acid Sequence↗
PubMed1989

Binding and transcription of relaxed DNA templates by fractions of maize chloroplast extracts.

Preparations of partially purified chloroplast DNA-dependent RNA polymerase from maize and some other plants transcribe cloned chloroplast genes preferentially and much more actively from appropriately negatively supercoiled templates than from relaxed templates. We have found that the polymerase in such fractions does not bind to promoter regions of the maize chloroplast genes psbA and rbcL on small linear DNA fragments but that some protein(s) in unfractionated chloroplast extracts does bind. DEAE chromatography of the extracts has permitted the separation of a DNA-binding fraction from the bulk of the RNA polymerase activity. The binding fraction contains plastid RNA polymerase activity that is relatively independent of template topology.

Amino Acid Sequence↗
PubMed1989

A 4-kDa maize chloroplast polypeptide associated with the cytochrome b6-f complex: subunit 5, encoded by the chloroplast petE gene.

Four polypeptides, three of which are chloroplast-encoded, have been shown to be associated with the thylakoid membrane cytochrome b6-f complex. In this report, the gene for a fifth polypeptide, which copurifies with the b6-f complex, is identified through the use of an antibody generated against a synthetic decapeptide predicted from a maize chloroplast DNA sequence. The deduced 37-amino acid sequence of the immunoreactive 4-kDa polypeptide is 100% and 86% conserved in the respective similar open reading frames encoded by Nicotiana tabacum and Marchantia chloroplast DNA. The 4-kDa polypeptide is present in both etioplasts and chloroplasts of maize and is found as well in spinach, tobacco, pea, wheat, and rice thylakoids. Similar to the other subunits of the b6-f complex, it is intrinsic to the membrane, and its hydrophilic COOH terminus is located at the stromal thylakoid surface. We propose to call the 4-kDa polypeptide "subunit 5" and the chloroplast gene that encodes it the petE gene.

Amino Acid Sequence↗
PubMed1989

Spectral Properties and Composition of Reaction Center and Ancillary Polypeptide Complexes of Photosystem II Deficient Mutants of Synechocystis 6803.

The polypeptide composition and spectral properties of three photosystem II (PSII) deficient mutants of the cyanobacterium Synechocystis 6803 have been determined. The levels of the 43 and 47 kilodalton chlorophyll-binding proteins and the reaction center component D2 are affected differently in each mutant; the 33 kD polypeptide of the oxygen-evolving complex is found at wild-type levels in all three. The 43 and 47 kilodalton proteins are implicated as important elements in the assembly and/or stability of the PSII reaction center, although the loss of one of these polypeptides does not lead to the loss of all PSII proteins. Low temperature fluorescence emission spectra of wild-type cells reveal chlorophyll-attributable peaks at 687 (PSII), 696 (PSII), and 725 (photosystem I) nanometers. All three mutants retain the 725 nanometer fluorescence but lack the 696 nanometer peak. This suggests that the latter fluorescence arises from PSII reaction center chlorophyll or results from interactions among functional PSII components in vivo. Cells that contain the 43 kilodalton and lack the 47 kilodalton protein, retain the 687 fluorescence; furthermore, in as much as this fluorescence is absent from cells without the 43 kilodalton protein, the 687 nanometer peak is judged to emanate from the 43 kilodalton chlorophyll-protein. A new peak, probably previously obscured, is revealed at 691 nanometers in cells that retain the 47 kilodalton protein but lack the 43 kilodalton polypeptide, suggesting that emission near 691 nanometers can be attributed to the 47 kilodalton polypeptide. Membrane-bound phycobilisomes are retained in these cells as is coupled-energy transfer between phycocyanin and allophycocyanin. Energy transfer to photosystem I by way of phycocyanin excitation proceeds as in wild-type cells despite the absence of certain PSII components.

Journal Article↗
PubMed1989

Studies on Chlamydomonas chloroplast transformation: foreign DNA can be stably maintained in the chromosome.

As shown originally by Boynton and co-workers (Boynton, J.E., Gillham, N.W., Harris, E.H., Hosler, J.P., Johnson, A.M., Jones, A.R., Randolph-Anderson, B.L., Robertson, D., Klein, T.M., Shark, K.B., and Sanford, J.C. [1988]. Science 240, 1534-1538), a nonphotosynthetic, acetate-requiring mutant strain of Chlamydomonas reinhardtii with a 2.5-kilobase pair deletion in the chloroplast Bam 10 restriction fragment region that removes the 3' half of the atpB gene and a portion of one inverted repeat can be transformed to photosynthetic competency following bombardment with microprojectiles coated with wild-type Bam 10 DNA. We have found that assorted other circular plasmids, single-strand DNA circles, or linear, duplex DNA molecules containing the wild-type atpB gene can also complement the same mutant. DNA gel blot hybridization analysis of all such transformants indicates that the complementing DNA has integrated into the chromosome at the atpB locus and suggests that a copy-correction mechanism operating between the inverted repeats maintains sequence identity in this region. Sequences from the intact inverted repeat may be recruited to restore the incomplete copy when exogenous DNA with only a portion of the deleted sequence is introduced. Furthermore, a foreign, unselected-for, chimeric gene flanked by chloroplast DNA sequences can be integrated and maintained stably in the chloroplast chromosome. The bacterial neomycin phosphotransferase structural gene fused to the maize chloroplast promoter for the large subunit gene of ribulose-1,5-biphosphate carboxylase (rbcL) has been integrated into the inverted repeat region of the Bam10 restriction fragment. RNA transcripts that hybridize to the introduced foreign gene have been identified.

Animals↗
PubMed1988

Thylakoid membrane protein topography: transmembrane orientation of the chloroplast cytochrome b-559 psbE gene product.

Protease accessibility and antibody to a COOH-terminal peptide were used as probes for the in situ topography of the Mr 10,000 psbE gene product (alpha subunit) of the chloroplast cytochrome b-559. Exposure of thylakoid membranes to trypsin or Staphylococcus aureus V8 protease cleaved the alpha subunit to a slightly smaller polypeptide (delta Mr approximately -1000) as detected on Western blots, without loss of reactivity to COOH-terminal antibody. The disappearance of the parent Mr 10,000 polypeptide from thylakoids in the presence of trypsin correlated with the appearance of the smaller polypeptide with delta Mr = -750, the conversion having a half-time of approximately 15 min. Exposure of inside-out vesicles to trypsin resulted in almost complete loss of reactivity to the antibody, showing that the COOH terminus is exposed on the lumenal side of the membrane. Removal of the extrinsic polypeptides of the oxygen-evolving complex resulted in an increase of the accessibility of the alpha subunit to trypsin. These data establish that the alpha subunit of cytochrome b-559 crosses the membrane once, as predicted from its single, 26-residue, hydrophobic domain. The NH2 terminus of the alpha polypeptide is on the stromal side of the membrane, where it is accessible, most likely at Arg-7 or Glu-6/Asp-11, to trypsin or V8 protease, respectively. As a consequence of this orientation, the single histidine residue in the alpha subunit is located on the stromal side of the hydrophobic domain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1988

Nuclear-organelle interactions: nuclear antisense gene inhibits ribulose bisphosphate carboxylase enzyme levels in transformed tobacco plants.

The biosynthesis of ribulose bisphosphate carboxylase (RUBISCO) provides a model system for studying the coordination of nuclear and organelle gene expression, since this abundantly transcribed and expressed chloroplast enzyme is composed of small (SS) and large subunits (LS) encoded by a nuclear multigene family and a single chloroplast gene, respectively. We have tested the possibility that SS mRNA or protein levels affect LS mRNA amounts or LS protein production and accumulation. We find that expression of antisense DNA sequences for the SS in transgenic tobacco plants drastically reduces the accumulation of SS mRNA and SS protein. These changes are accompanied by corresponding reductions of LS protein but not LS mRNA amounts; accumulation of the LS protein appears to be regulated by translational and posttranslational factors. We also find that the transgenic plants display striking variations in growth that are correlated with antisense gene dosage.

Cell Nucleus↗
PubMed1988

Yeast activators stimulate plant gene expression.

GAL4 is a transcriptional activator found in yeast. Two distinct functions of the protein are required for its activity: one directs sequence-specific DNA binding, and another interacts with some other component of the transcriptional machinery, for example, RNA polymerase II or a TATA-binding protein. Two short regions of GAL4 function as 'activating sequences' when attached to the DNA-binding portion of GAL4 and these regions can be replaced by a large number of peptides encoded by Escherichia coli genomic DNA fragments or by a synthetic peptide designed to form an amphiphilic alpha-helix. All of these activating sequences, like that found in another yeast activator, GCN4 bear an excess negative charge. GAL4 and its derivatives that are active in yeast stimulate transcription in mammalian cells when GAL4 binding sites are introduced upstream of a mammalian gene; similarly, GAL4 activates transcription in Drosophila cells. Here we show that GAL4 derivatives stimulate gene expression in plant cells.

Acetyltransferases↗
PubMed1988

Chloroplast DNA replication in vitro: site-specific initiation from preferred templates.

An enzyme system prepared from maize chloroplasts catalyzes the synthesis of DNA from maize chloroplast DNA sequences cloned in bacterial plasmids. Cloned maize chloroplast DNA fragments Bam HI 17' (2470 bp) and Eco RI x (1368 bp) have been shown to be preferred templates for in vitro DNA synthesis catalyzed by pea chloroplast DNA polymerase preparations [Gold et al. (1987) Proc. Natl. Acad. Sci. USA 84, 194-198]. Analysis of replicative intermediates indicates that although the template activity of the recombinant plasmid pZmcBam 17' is substantially greater than that of the pZmcEco x, replication in both cases originates from within a 455 bp region which overlaps the two plasmids. The remaining approximately 1500 basepair portion of maize chloroplast BamHI fragment 17' is not more active because it contains additional origins for replication. The overlapping region shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that contains a replication origin. Replication is shown to proceed bidirectionally within the 455 bp origin region. Recombinant plasmid pZmc 427, which is also active in the in vitro DNA synthesis assay, promoted localized replication initiation within a 1 kbp Bg1II-Eco RI fragment of the chloroplast DNA insert, a region that includes the 3' terminal part of the psbA gene.

Chloroplasts↗
PubMed1988

Molecular analysis of a mutant defective in photosynthetic oxygen evolution and isolation of a complementing clone by a novel screening procedure.

Photosynthesis-defective mutants of the transformable cyanobacterium Synechocystis 6803 have been isolated following nitrosoguanidine mutagenesis. The photosystem II- phenotype of one of these mutants is shown by DNA sequencing to be attributable to a short deletion in psbC, the gene encoding the 44-kd, chlorophyll-binding protein of photosystem II. Although not a component of the reaction center of photosystem II, the 44-kd protein is none the less shown to be essential in vivo for photosystem II activity. The deletion in psbC also results in greatly diminished levels of D-2 (a component of the reaction center of photosystem II) indicating that the loss of the product of the psbC gene affects the assembly or stability of the photosystem II reaction center. The isolation of a clone capable of restoring both photosystem II activity and photoautotrophy to the mutant cells was aided by the observation that restriction fragments or cloned Synechocystis 6803 DNA applied in liquid or in melted agarose directly onto a lawn of Synechocystis 6803 will lead to the transformation of the cells. This in situ 'dot' transformation procedure provides a convenient method for the rapid identification of fractions or clones containing complementing Synechocystis 6803 DNA.

Amino Acid Sequence↗
PubMed1988

Relocating a gene for herbicide tolerance: A chloroplast gene is converted into a nuclear gene.

The chloroplast gene psbA codes for the photosynthetic quinone-binding membrane protein Q(B), which is the target of the herbicide atrazine. This gene has been converted into a nuclear gene. The psbA gene from an atrazine-resistant biotype of Amaranthus hybridus has been modified by fusing its coding region to transcription-regulation and transit-peptide-encoding sequences of a bona fide nuclear gene. The constructs were introduced into the nuclear genome of tobacco by using the Agrobacterium tumor-inducing (Ti) plasmid system, and the protein product of nuclear psbA has been identified in the photosynthetic membranes of chloroplasts. Recovery of atrazine-tolerant transgenic plants shows that the product of the transplanted gene functions in photosynthesis. These experiments show that it is possible to modify chloroplast-gene-specified functions via nuclear-genome transformation and also raise evolutionary questions.

Journal Article↗
PubMed1988

Differential Expression in Bundle Sheath and Mesophyll Cells of Maize of Genes for Photosystem II Components Encoded by the Plastid Genome.

We have explored the cell-specific expression of plastid genes encoding five photosystem II proteins (PSII-A, -B, -C, -D and cytochrome (Cyt) b-559 encoded by plastid genes psbA, B, C, D and E plus F, respectively) and one encoding the photosystem I protein PSI-A1 in bundle sheath and mesophyll cells (BSC and MC) at different stages of photoregulated development in leaves of dark-grown maize seedlings. Two types of cell-specific mRNA ccumulation patterns have been discerned. First is the PSII-A type: the mRNA is present in small but about equal amounts in both cell types in etiolated leaves; then, upon illumination, the level of mRNA increases progressively and remains high in MC but not BSC of green leaves. Second is the Cyt b-559 type: MC of etiolated leaves have about twice as much of this mRNA as do BSC; about a twofold to threefold transient increase is induced by light in MC but not in BSC. The genes for the PSII-C and -D proteins are complementary to a sizable family of transcripts over a large range of sizes; the patterns of changes in pools of some of these transcripts are of the PSII-A type and others are of the Cyt b-559 type. The DNA region encoding the PSII-B protein also hybridizes to multiple transcripts; among them is a prominent 2.2 kilobase transcript that follows the Cyt b-559 accumulation pattern. In contrast, the pattern of PSI-A1 mRNA accumulation is the same in both cell types. Accumulation of the PSII-B and -D proteins is light-dependent and coordinate. Both are much more abundant in MC than in BSC. PSII-A is detectable in MC of etiolated leaves and increases during photoregulated chloroplast maturation in both cell types, however, to a much greater extent in MC. We conclude that the expression of each of these plastidencoded photosystem II genes in BSC and MC is photoregulated differently; both transcript abundance and translation are regulated.

Journal Article↗
PubMed1988

Diurnal and Circadian Rhythms in the Accumulation and Synthesis of mRNA for the Light-Harvesting Chlorophyll a/b-Binding Protein in Tobacco.

The steady state level of mRNA for the light-harvesting chlorophyll a/b binding protein (LHCP) varies considerably during a day-night light cycle in expanding leaves of tobacco (Nicotiana tabacum, cv Wisconsin 38). The maximum (day) level is at least one hundred-fold higher than the minimum (night) level. The oscillation in the LHCP mRNA level continues for several days even under constant illumination. However, no such circadian rhythm can be detected in constant darkness. Plants that have been maintained in constant darkness for a prolonged period reinitiate the circadian rhythm upon illumination: the LHCP mRNA level resumes its rhythmic behavior with a period of roughly 24 hours. The same circadian fluctuation of LHCP mRNA is observed upon illuminating etiolated tobacco seedlings, demonstrating that the circadian rhythm is not dependent on the presence of mature chloroplasts. In vitro transcription experiments with nuclei isolated from green plants during various times of the day indicate that the diurnal as well as the circadian changes of the level of LHCP RNA are due, at least in part, to changes in the rate of LHCP mRNA synthesis.

Journal Article↗
PubMed1987

Nucleotide sequence, promoter analysis, and linkage mapping of the unusually organized operon encoding ribosomal proteins S7 and S12 in maize chloroplast.

The nucleotide sequence of the operon encoding maize chloroplast ribosomal protein genes S7 and S12 and the promoter activity of a chimeric construct of the -10/-35 sequence of this operon (attached to a promoterless chloramphenicol acetyltransferase gene) have been determined. This operon occurs in the chloroplast genome divided in two parts: part A contains exon 1 of rpS12 (encoding the N-terminal 38 amino acid residues), whereas part B has the following structure: promoter-rpS12 (exon 2 + intron + exon 3)-spacer-rpS7-terminator. Part A is located at the approximate coordinate position 41000, whereas two copies of part B are located at two distant locations in the genome at coordinate positions 18700 and 120200. This unusual organization of the S12 operon in maize (a monocot plant) is similar to that reported in a dicot and a lower plant. The deduced amino acid sequence of maize chloroplast S7 shows 43, 38, 71, and 85% and of S12 shows 66, 72, 91 and 90% sequence identity to the corresponding sequences of Escherichia coli, Euglena gracilis, Marchantia polymorpha, and Nicotiana tabacum, respectively. The promoter upstream of rpS12 (part B) is transcriptionally active in E. coli.

Amino Acid Sequence↗
PubMed1987

Differential expression of C4 pathway genes in mesophyll and bundle sheath cells of greening maize leaves.

Pyruvate orthophosphate dikinase, phosphoenolpyruvate carboxylase, and NADP-malate dehydrogenase function in a series of reactions for fixing CO2 in mesophyll cells and NADP-malic enzyme (ME) catalyzes the production of CO2 and NADPH in bundle sheath cells of maize which is a NADP-ME type C4 plant. Northern blot analyses with cDNA clones for pyruvate orthophosphate dikinase and phosphoenolpyruvate carboxylase and in vitro translation-immunoprecipitation experiments with antiserum to NADP-malate dehydrogenase showed that pools of transcripts of these three genes grow and shrink coordinately in mesophyll cells but not in bundle sheath cells upon illumination of dark-grown maize seedlings. Western blot analyses indicated that the protein levels of phosphoenolpyruvate carboxylase and pyruvate orthophosphate dikinase are low in dark-grown maize seedlings and increase progressively following light-induced transient accumulation of their mRNAs in mesophyll cells. These proteins continue to accumulate and plateau in late-greening and green leaves in spite of a rapid drop in the sizes of their mRNA pools. Surprisingly, relatively large amounts of NADP-malate dehydrogenase are present in mesophyll cells of etiolated leaves despite the low level of the corresponding mRNA. No phosphoenolpyruvate carboxylase or NADP-malate dehydrogenase were detected in bundle sheath cells. On the other hand, the ME gene responds to light induction at both the transcriptional and translational levels only in bundle sheath cells. Moreover, the steady-state level of ME mRNA stays high in late-greening and green leaves in contrast to the rapid decline of mRNA levels of three other C4 pathway genes in mesophyll cells. In addition, low levels of both the mRNA and protein encoded by the PPDK gene were detected in bundle sheath cells. These levels were not influenced by light as distinguished from the patterns observed in mesophyll cells.

Centrifugation, Density Gradient↗