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At least 19 recordsLinked to original sources

PubMed1977

Cellular content of chloroplast DNA and chloroplast ribosomal RNA genes in Euglena gracilis during chloroplast development.

The cellular content of chloroplast DNA in Euglena gracilis has been quantitatively determined. DNA was extracted from Euglena cells at various stages of chloroplast development and renatured in the presence of trace amounts of 3H-labeled chloroplast DNA. From the kinetics of renaturation of the 3H-labeled chloroplast DNA, compared with the kinetics of renaturation of excess nonradioactive chloroplast DNA, the fraction of cellular DNA represented by chloroplast DNA was calculated. The content of chloroplast DNA was found to increase from 4.9 to 14.6% of cellular DNA during light-induced chloroplast development. Correcting for the change in DNA mass per cell, the number of copies of chloroplast DNA is found to vary from 1400 to 2900 per cell. During this developmental transition, the cellular content of the chloroplast ribosomal RNA genes varies from 1900 to 5200 copies per cell. The ratio of the number of copies of rRNA genes to chloroplast genomes per cell remains in the range of 1-2 throughout chloroplast development, ruling out selective amplification of chloroplast rRNA genes as a means of regulation of rRNA gene expression. Direct measurement of the number of rRNA cistrons per 9.2 X 10(7) dalton genome yields a value of 1 or 2.

Animals↗

PubMed1980

Hybridization of bean, spinach, maize and Euglena chloroplast transfer RNAs with homologous and heterologous chloroplast DNAs. An approach to the study of homology between chloroplast tRNAs from various species.

Chloroplast tRNAs from two dicotyledons (spinach and bean), a monocotyledon (maize) and a green alga (Euglena) have been fractionated by two-dimensional gel electrophoresis. The individual tRNAs have been identified, albeled with 125I or 32P, and used in tRNA-DNA hybridization experiments. Spinach chloroplast tRNAs hybridize as well, and maize chloroplast tRNAs almost as well as bean chloroplast tRNAs to bean chloroplast DNA, thus suggesting a high degree of homology between the chloroplast tRNAs from the two dicotyledons and between the tRNAs from the two dicotyledons and those of the monocotyledon. But Euglena total chloroplast tRNA hybridizes very poorly to bean chloroplast DNA, and among the 14 individual tRNAs tested, only one, Euglena chloroplast tRNAPhe, hybridizes to both maize and bean chloroplast DNAs, which is in good agreement with the fact that Euglena and bean chloroplast tRNAsPhe have almost identical primary structures.

Amino Acids↗

PubMed2002

A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts.

We generated transgenic tobacco (Nicotiana tabacum cv Xanthi) plants that contained only one to three enlarged chloroplasts per leaf mesophyll cell by introducing NtFtsZ1-2, a cDNA for plastid division. These plants were used to investigate the advantages of having a large population of small chloroplasts rather than a few enlarged chloroplasts in a leaf mesophyll cell. Despite the similarities in photosynthetic components and ultrastructure of photosynthetic machinery between wild-type and transgenic plants, the overall growth of transgenic plants under low- and high-light conditions was retarded. In wild-type plants, the chloroplasts moved toward the face position under low light and toward the profile position under high-light conditions. However, chloroplast rearrangement in transgenic plants in response to light conditions was not evident. In addition, transgenic plant leaves showed greatly diminished changes in leaf transmittance values under both light conditions, indicating that chloroplast rearrangement was severely retarded. Therefore, under low-light conditions the incomplete face position of the enlarged chloroplasts results in decreased absorbance of light energy. This, in turn, reduces plant growth. Under high-light conditions, the amount of absorbed light exceeds the photosynthetic utilization capacity due to the incomplete profile position of the enlarged chloroplasts, resulting in photodamage to the photosynthetic machinery, and decreased growth. The presence of a large number of small and/or rapidly moving chloroplasts in the cells of higher land plants permits more effective chloroplast phototaxis and, hence, allows more efficient utilization of low-incident photon flux densities. The photosynthetic apparatus is, consequently, protected from damage under high-incident photon flux densities.

Biological Transport↗

PubMed1975

Biogenesis of chloroplast membranes in Chlamydomonas reinhardi: Chloroplast-controlled transfer of cytoplasmic proteins to the developing chloroplast membranes as visualized by quantitative radioautography.

The light-induced formation of the photosynthetic membranes (greening) in y-1 mutant of Chlamydomonas reinhardi requires synthesis of new proteins which become incorporated into the growing membranes. It has been shown previously (Eytan and Ohad, 1970) that proteins synthesized by both chloroplast and cytoplasmic ribosomes concur in the formation of functional photosynthetic membranes, indicating the presence of a mechanism permitting the specific transfer of membrane proteins synthesized in the cytoplasm into the chloroplast. Transfer of such proteins cannot yet be identified by the usual biochemical techniques unless they become part of the growing photosynthetic membranes. However, it is possible to follow their synthesis and translocation between the different cellular compartments by use of quantitative electron microscopic radioautography. In the present work, the radioautographic grain distribution among chloroplast, chloroplast membrane, nucleus, mitochondria, and the remainder of the cytoplasm (cytosol) was carried out following short radioactive pulse-labeling and chase during greening of dark-grown mutants in the presence or absence of protein synthesis inhibitors. The results indicate that transport of some of the proteins of cytoplasmic origin to their final location within the chloroplast is at least partially controlled by concomitant synthesis of proteins by the chloroplast ribosomes.

Arginine↗

PubMed1979

Light-Dark Regulation of Starch Metabolism in Chloroplasts: II. Effect of Chloroplastic Metabolite Levels on the Formation of ADP-Glucose by Chloroplast Extracts.

The rate of ADP-glucose formation from [(14)C]glucose 6-phosphate and ATP by the soluble fraction of lysed chloroplasts is studied as a function of the levels of metabolites (3-phosphoglycerate, orthophosphate, hexose monophosphate, and ATP) as determined in whole chloroplasts of Spinacia oleracea in light and dark.A change in 3-phosphoglycerate concentration (from 4 to 1.4 millimolar, as in whole chloroplasts during light-dark transition) decreases the rate of ADP-glucose formation 6- to 7-fold. An increase in hexose monophosphate concentration from 2 to 6 millimolar, which occurs at the same time in whole chloroplasts, stimulates ADP-glucose formation only slightly.At constant levels of orthophosphate (4 millimolar) and 3-phosphoglycerate (4 millimolar), a change in ATP concentration from 0.2 to 1 millimolar causes an immediate 4- to 5-fold increase in the rate of ADP-glucose formation. Another significant stimulation of ADP-glucose formation (about 4- to 6-fold) is obtained after addition of dithiothreitol at high concentrations (50 millimolar). A simultaneous increase in the concentrations of 3-phosphoglycerate, ATP, and dithiothreitol, with orthophosphate and Mg(2+) being constant at 4 and 5 millimolar, respectively, causes a 130-fold increase in the rate of ADP-glucose formation (from 0.042 to 5.49 microgram atoms carbon per milligram chlorophyll per hour).The role of these and other factors is discussed with respect to light-dark regulation of starch formation in intact chloroplasts.

Journal Article↗

PubMed1980

Influence of Hydrogen Peroxide upon Carbon Dioxide Photoassimilation in the Spinach Chloroplast: I. HYDROGEN PEROXIDE GENERATED BY BROKEN CHLOROPLASTS IN AN "INTACT" CHLOROPLAST PREPARATION IS A CAUSAL AGENT OF THE WARBURG EFFECT.

Photosynthesis and the Warburg effect (O(2) inhibition of photosynthesis) were evaluated in preparations of intact spinach chloroplasts enriched with varying amounts of lysed chloroplasts. Increasing the ratio of broken to intact plastids resulted in decreased rates of CO(2) assimilation.Hydrogen peroxide when added at 10 or more micromolar also inhibited photosynthesis in these preparations. Inhibition of the photosynthetic rate by both factors was eliminated by addition of catalase. These findings indicate that H(2)O(2) presumably generated by the broken chloroplasts was the causal agent of this inhibition.The Warburg effect also became more pronounced by increasing the level of broken to intact chloroplasts. This effect was examined as a function of added catalase, pH, and O(2) concentration. At 21% O(2) and 0.44 to 0.68 millimolar CO(2), catalase relieved the effect almost completely at pH 7.5, but at pH 8.3, the rate was restored only to about half or less of the control. At pH 7.6, 0.44 millimolar CO(2), and 100% O(2), the effect was only slightly overcome by catalase.A rise in glycolate synthesis by the isolated spinach chloroplast has been shown previously to be coupled to an increase in pH and O(2) (Kow, Robinson, Gibbs 1977 Plant Physiol 60: 492-495; Robinson, Gibbs, Cotler 1977 Plant Physiol 59: 530-534). At 21% O(2), glycolate synthesis was not affected by the addition of catalase at pH 7.5 or 8.3. It is proposed that at 21% O(2) and without some means of removing H(2)O(2), that portion of the Warburg effect attributed to glycolate synthesis has been overestimated at pH values in the order of 7.5. In contrast, that portion of the Warburg effect which was, at alkaline pH, insensitive to catalase represented the stress placed upon the photosynthetic carbon reduction cycle which resulted from an enhanced synthesis of glycolate. At 100% O(2) aeration and pH 7.5 to 8.5, the Warburg effect may also represent O(2)-mediated inhibition of a Calvin cycle enzyme within the intact plastid.

Journal Article↗

PubMed1991

Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA-binding domains.

Two new ribonucleoproteins (RNPs) have been identified from a tobacco chloroplast lysate. These two proteins (cp29A and cp29B) are nuclear-encoded and have a less affinity to single-stranded DNA as compared with three other chloroplast RNPs (cp28, cp31 and cp33) previously isolated. DNA sequencing revealed that both contain two consensus sequence-type homologous RNA-binding domains (CS-RBDs) and a very acidic amino-terminal domain but shorter than that of cp28, cp31 and cp33. Comparison of cp29A and cp29B showed a 19 amino acid insertion in the region separating the two CS-RBDs in cp29B. This insertion results in three tandem repeats of a glycine-rich sequence of 10 amino acids, which is a novel feature in RNPs. The two proteins are encoded by different single nuclear genes and no alternatively spliced transcripts could be identified. We constructed a phylogenetic tree for the ten chloroplast CS-RBDs. These results suggest that there is a sizable RNP family in chloroplasts and the diversity was mainly generated through a series of gene duplications rather than through alternative pre-mRNA splicing. The gene for cp29B contains three introns. The first and second introns interrupt the first CS-RBD and the third intron does the second CS-RBD. The position of the first intron site is the same as that in the human hnRNP A1 protein gene.

Amino Acid Sequence↗

PubMed1982

Transport of proteins into chloroplasts. Binding of nuclear-coded chloroplast proteins to the chloroplast envelope.

A system has been constructed in vitro for the binding of cytoplasmically synthesized chloroplast proteins to the chloroplast envelope which precedes the uptake into the organelle in vivo. Isolated chloroplast envelopes from young pea or spinach are capable of binding the majority of proteins obtained by translation of poly(A)-containing RNA from greening plants in vitro. Among the bound proteins the precursors to the light-harvesting chlorophyll a/b apoprotein and the small subunit of ribulose-1,5-bisphosphate carboxylase are prominent. Binding is an intrinsic property of the envelope membrane and does not require energy in the form of ATP. Bound proteins remain on the surface of the envelope vesicles and can be digested by protease. Binding is complete within minutes, shows a high affinity of the reactants, and is non-ionic in nature. Protein binding is specific for translation products of poly(A)-containing RNA from greening plants. Precursors to chloroplast protein are bound preferentially as compared to the mature proteins. The specificity is further demonstrated by the low binding of proteins obtained by run-off translation of polysomes. Binding of radioactive labeled proteins is subject to competition by excess unlabeled homologous proteins. Once bound, the proteins are withdrawn from competition indicating a high binding stability. All the properties found for binding of proteins to isolated envelopes are consistent with the concept of the so-called envelope carrier hypothesis.

Binding, Competitive↗

PubMed1980

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates.

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light-dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development.

Chlamydomonas↗

PubMed1985

Osmotic adjustment by intact isolated chloroplasts in response to osmotic stress and its effect on photosynthesis and chloroplast volume.

Spinach leaf chloroplasts isolated in isotonic media (330 millimolar sorbitol, -1.0 megapascals osmotic potential) had optimum rates of photosynthesis when assayed at -1.0 megapascals. When chloroplasts were isolated in hypertonic media (720 millimolar sorbitol, -2.0 megapascals osmotic potential) the optimum osmotic potential for photosynthesis was shifted to -1.8 megapascals and the chloroplasts had higher rates of CO(2)-dependent O(2) evolution than chloroplasts isolated in 330 millimolar sorbitol when both were assayed at high solute concentrations.Transfer of chloroplasts isolated in 330 millimolar sorbitol to 720 millimolar sorbitol resulted in decreased chloroplast volume but this shrinkage was only transient and the chloroplasts subsequently swelled so that within 2 to 3 minutes at 20 degrees C the chloroplast volume had returned to near the original value. Thus, actual steady state chloroplast volume was not decreased in hypertonic media. In isotonic media, there was a slow but significant uptake of sorbitol by chloroplasts (10 to 20 micromoles per milligram chlorophyll per hour at 20 degrees C). Transfer of chloroplasts from 330 millimolar sorbitol to 720 millimolar sorbitol resulted in rapid uptake of sorbitol (up to 280 micromoles per milligram chlorophyll per hour at 20 degrees C) and after 5 minutes the concentration of sorbitol inside the chloroplasts exceeded 500 millimolar. This uptake of sorbitol resulted in a significant underestimation of chloroplast volume unless [(14)C]sorbitol was added just prior to centrifuging the chloroplasts through silicone oil. Sudden exposure to osmotic stress apparently induced a transient change in the permeability of the chloroplast envelope since addition of [(14)C]sorbitol 3 minutes after transfer to hypertonic media (when chloroplast volume had returned to normal) did not result in rapid uptake of labeled sorbitol.It is concluded that chloroplasts can osmotically adjust in vitro by uptake of solutes which do not normally penetrate the chloroplast envelope, resulting in a restoration of normal chloroplast volume and partially preventing the inhibition of photosynthesis by high solute concentrations. The results indicate the importance of matching the osmotic potential of isolation media to that of the tissue, particularly in studies of stress physiology.

Journal Article↗

PubMed1979

The route of entry of cytoplasmically synthesized proteins into chloroplasts of algae possessing chloroplast ER.

In 8 classes of algae, namely the Cryptophyceae, Raphidophyceae, Haptophyceae, Chrysophyceae, Bacillariophyceae, Xanthophyceae, Eustigmatophyceae and Phaeophyceae, the chloroplasts, in addition to being surrounded by a double-membraned chloroplast envelope, are also enclosed by a cisterna of endoplasmic reticulum called the chloroplast ER. Often this ER cisterna is continuous with the outher membrane of the nuclear envelope in such a manner that the nuclear envelope forms a part of the ER sac enclosing the chloroplast. In all these classes of algae except the Cryptophyceae, a regular network of tubules and vesicles, named the periplastidal reticulum, is present at a specific location between the chloroplast envelope and the chloroplast ER. In the Cryptophyceae, scattered vesicles are found between the chloroplast envelope and the chloroplast ER. Ribosomes which have been shown to be arranged to polysomes are found on the outer membrane of the chloroplast ER. It is proposed that nuclear-coded proteins which are destined for the chloroplast are synthesized on these polysomes, passing during synthesis into the lumen of the ER cisterna. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles containing these proteins then pinch off the chloroplast ER and form the periplastidal reticulum. Vesicles then fuse with the outer membrane of the chloroplast envelope thereby delivering their contents to the lumen of the chloroplast envelope. Proteins then cross the inner membrane of the chloroplast envelope in an as yet unknown manner. Experimental evidence for this hypothesis comes from studies on Ochromonas danica using chloramphenicol and spectinomycin, which inhibit protein synthesis on plastid ribosomes, and cycloheximide, which inhibits protein synthesis on cytoplasmic ribosomes. In cells of Ochromonas exposed to chloramphenicol or spectinomycin, the periplastidal reticulum proliferates markedly becoming several layers thick. Presumably this build up of periplastidal reticulum occurs because the transport of cytoplasmically synthesized plastid proteins is slowed down when protein synthesis in the chloroplast is inhibited. Conversely, when cells of Ochromonas are treated with cycloheximide, there is a reduction in the amount of periplastidal reticulum presumably because there are no cytoplasmically synthesized proteins to be transported into the chloroplast.

Biological Transport↗

PubMed1976

A measurement of the fraction of chloroplast DNA transcribed during chloroplast development in Euglena gracilis.

The fraction of chloroplast DNA transcribed at different stages of chloroplast development in Euglena gracilis was measured by RNA-DNA hybridization. Euglena cells were grown in the dark in a heterotrophic medium to stationary phase and then transferred to the light. Chloroplast development was monitored by the increase in the cellular chlorophyll content in the absence of cell division. Total cell RNA was isolated at various stages of chloroplast development, and hybridized in a vast excess to [125I]chloroplast DNA. The fraction of [125I]chloroplast DNA in the form of a duplex was monitored by chromatography on hydroxylapatite columns. The amount of RNA-DNA hybrid in the duplex mixture was determined by correcting for the contribution of DNA-DNA renaturation under the same conditions. The fraction of chloroplast DNA transcribed was calculated by multiplying by two the amount of single-stranded DNA in the form of an RNA-DNA hybrid. Prior to the initiation of chloroplast development (i.e., in dark grown cells) the fraction of chloroplast DNA represented as RNA transcripts in the cell is 0.53. As chloroplast development proceeds, the fraction of the chloroplast DNA transcribed decreases to 0.47. Experiments in which mixtures of various RNA samples were hybridized to the chloroplast DNA indicate that there is a small portion of chloroplast DNA transcribed at later stages of chloroplast development which is not represented as transcripts at the onset of chloroplast development. Melting properties of the RNA-DNA hybrids show that the RNA-DNA duplexes are slightly less stable than renatured [125I]chloroplast DNA.

Animals↗

PubMed2005

Homologous and heterologous reconstitution of Golgi to chloroplast transport and protein import into the complex chloroplasts of Euglena.

Euglena complex chloroplasts evolved through secondary endosymbiosis between a phagotrophic trypanosome host and eukaryotic algal endosymbiont. Cytoplasmically synthesized chloroplast proteins are transported in vesicles as integral membrane proteins from the ER to the Golgi apparatus to the Euglena chloroplast. Euglena chloroplast preprotein pre-sequences contain a functional N-terminal ER-targeting signal peptide and a domain having characteristics of a higher plant chloroplast targeting transit peptide, which contains a hydrophobic stop-transfer membrane anchor sequence that anchors the precursor in the vesicle membrane. Pulse-chase subcellular fractionation studies showed that (35)S-labeled precursor to the light harvesting chlorophyll a/b binding protein accumulated in the Golgi apparatus of Euglena incubated at 15 degrees C and transport to the chloroplast resumed after transfer to 26 degrees C. Transport of the (35)S-labeled precursor to the chlorophyll a/b binding protein from Euglena Golgi membranes to Euglena chloroplasts and import into chloroplasts was reconstituted using Golgi membranes isolated from 15 degrees C cells returned to 26 degrees C. Transport was dependent upon extra- and intrachloroplast ATP and GTP hydrolysis. Golgi to chloroplast transport was not inhibited by N-ethylmaleimide indicating that fusion of Golgi vesicles to the chloroplast envelope does not require N-ethylmaleimide-sensitive factor (NSF). This suggests that N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are not utilized in the targeting fusion reaction. The Euglena precursor to the chloroplast-localized small subunit of ribulose-1,5-bisphosphate carboxylase was not imported into isolated pea chloroplasts. A precursor with the N-terminal signal peptide deleted was imported, indicating that the Euglena pre-sequence has a transit peptide that functions in pea chloroplasts. A precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase with the hydrophobic membrane anchor and the pre-sequence region C-terminal to the hydrophobic membrane anchor deleted was imported localizing the functional transit peptide to the Euglena pre-sequence region between the signal peptidase cleavage site and the hydrophobic membrane anchor. The Euglena precursor to the small subunit of ribulose-1,5-bisphosphate carboxylase and the deletion constructs were not post-translationally imported into isolated Euglena chloroplasts indicating that vesicular transport is the obligate import mechanism. Taken together, these studies suggest that protein import into complex Euglena chloroplasts evolved by developing a novel vesicle fusion targeting system to link the host secretory system to the transit peptide-dependent chloroplast protein import system of the endosymbiont.

Adenosine Triphosphate↗

PubMed2002

Searching limiting steps in the expression of chloroplast-encoded proteins: relations between gene copy number, transcription, transcript abundance and translation rate in the chloroplast of Chlamydomonas reinhardtii.

We performed a systematic investigation of the quantitative relationship between genome copy number, transcription, transcript abundance and synthesis of photosynthetic proteins in the chloroplast of the green algae Chlamydomonas reinhardtii grown either in mixotrophic or phototrophic conditions. The chloroplast gene copy number is lower in the latter condition and the half-life and accumulation levels of most chloroplast transcripts are significantly reduced, although the relative rates of protein synthesis remain similar. Our study shows that, in most instances, chloroplast protein synthesis is poorly sensitive to changes in gene copy number or transcript abundance in the chloroplast. Treatment with 5-fluoro-2'-deoxyuridine, that inhibits chloroplast DNA replication and decreases extensively the number of copies of the chloroplast genome, had limited effects on the abundance of most chloroplast transcripts and little if any effect on the rates of protein synthesis. When using rifampicin, that selectively inhibits chloroplast transcription, we found no direct correlation between the level of transcripts remaining in the chloroplast and the rates of chloroplast protein synthesis. For two chloroplast genes, a 90% decrease in the amount of transcript did not cause a drop in the rate of synthesis of the corresponding protein product. Overall, our results demonstrate that there is no gene dosage effect in the chloroplast and that transcript abundance is not limiting in the expression of chloroplast-encoded protein.

Animals↗

PubMed1980

Photomorphogenic Regulation of Chloroplast Replication in Euglena: ENHANCED LOSS OF CHLOROPLAST DNA IN RED LIGHT.

Chloroplast replication in Euglena gracilis is specifically inhibited by ultraviolet light and the effect is photoreactivable.The ability of irradiated cells to be photoreactivated is lost more rapidly if cells are incubated in red light than in darkness. A mutant, Y(9)ZNa1L, which lacks the red-blue photomorphogenic system regulating chloroplast synthesis does not show the red-light-enhanced loss of photoreactivability. Another mutant, Y(11)P(27)ZD which has the red-blue system, but lacks the blue-light system also regulating chloroplast synthesis, shows the red-light effect. The red-light effect is seen in a mutant of photosynthetic electron transport, P(4)ZUL, which rules out a product of photosynthesis as a mediator of the effect. Inhibitors of protein synthesis on chloroplast ribosomes do not prevent the red-light-enhanced loss of chloroplast DNA. Chloroplast DNA is lost rapidly when UV-irradiated cells are incubated in red light, showing that the loss of photoreactivability is due to the loss of the substrate for photoreactivation, chloroplast DNA. Therefore, the red-blue photomorphogenic system is activating a chloroplast DNA-specific nuclease(s). A model is proposed for a light-mediated mechanism regulating the amount of chloroplast DNA: blue light would promote chloroplast DNA synthesis; red light would promote its degradation. The photomorphogenic systems regulating chloroplast synthesis might work by activating a chloroplast-specific modification-restriction mechanism.

Journal Article↗

PubMed1976

Changes in the expression of the chloroplast genome of Euglena gracilis during chloroplast development.

The transcription program from the chloroplast genome of Euglena gracilis Z during light-induced chloroplast development has been characterized by hybridization of total cell RNA to 3H-labeled chloroplast DNA. Pancreatic DNase activated, purified Euglena chloroplast DNA was enzymatically labeled by Escherichia coli DNA polymerase I with [3H]TTP as a substrate. The [3H]DNA 'hybridization probe" was characterized by the kinetics of its renaturation with purified chloroplast DNA, and the thermal stability of [3H]DNA-DNA, and [3H]DNA-RNA hybrids. The [3H]DNA was hybridized in trace amounts to total cellular RNA extracted from Euglena cells 0, 4, 8, 12, 24, 48, and 72 h after the onset of chloroplast development. A large percentage (17%) of the chloroplast genome was found to be transcribed in dark adapted cells. Development is marked by an initial decrease in the fraction of the genome transcribed followed by an increase to 23% transcribed at the end of 72 h of light growth. Chloroplast RNA transcripts were also characterized by the kinetics of their hybridization to chloroplast DNA. The chloroplast specific RNA population is composed of three abundance classes, and the R0t1/2 for each class varies during the early stages of chloroplast development.

Chlorophyll↗

PubMed1985

In vitro expression of chloroplast genes in lysates of higher plant chloroplasts.

A DNA-dependent in vitro-coupled transcription-translation system has been prepared from lysates of isolated chloroplasts. These lysates are comparable to those of Escherichia coli in transcriptional and translational fidelity and efficiency in response to a given template DNA. When Nicotiana tabacum chloroplast DNA is used as template with chloroplast lysates (N. tabacum or spinach) or E. coli lysates, NaDodSO4 gel analysis reveals similar polypeptide patterns that are distinct from the patterns obtained with E. coli DNA. Genes in recombinant plasmids containing chloroplast DNA are also expressed in these in vitro systems. DNA . RNA hybridization experiments show that transcripts are synthesized from most of the chloroplast genome. Newly synthesized large subunit of ribulosebisphosphate carboxylase/oxygenate and a transcript of the large subunit gene (rbcL) are observed in chloroplast lysates using as template chloroplast DNA or cloned fragments of tobacco chloroplast DNA that contain the large subunit gene. Results suggest that differential expression of chloroplast genes occurs in vitro. By using cloned chloroplast DNA templates in this homologous system, it is possible to identify and map structural genes for chloroplast proteins.

Cell-Free System↗

PubMed1990

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Cells, Cultured↗