Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Chloroplasts”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

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↗
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↗
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↗
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↗
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↗
PubMed1979

The association of chloroplast DNA with photosynthetic membrane vesicles from spinach chloroplasts.

To investigate the association between chloroplast DNA (cp DNA) and the photosynthetic membranes of spinach chloroplasts, previously suggested by electron-microscope autoradiography, use has been made of vesicles formed by isolating chloroplasts directly in 3.5 mM Mg2+. These chloroplast vesicles consist of photosynthetic membranes, separate from chloroplast envelope membranes. Light and electron microscopy confirm that the vesicles consist of swollen stroma lamellar membranes with some peripheral grana lamellae that are much less swollen. Vesicles labelled with [H]thymidine were obtained from [3H]thymidine-labelled chloroplasts from spinach disks in which chloroplast division and cp DNA synthesis and segregation were occurring. The chloroplast vesicle fraction retains about 45% of the cp DNA as determined by liquid scintillation counting. The cp DNA-membrane associations do not appear to be dependent on the presence of Mg2+. The chloroplast vesicles can be autoradiographed for light microscopy if they are fixed in formaldehyde and no centrifugation steps are used. Light-microscope autoradiography is consistent with a preferential labelling of grana as opposed to stroma membranes, and long lengths of membrane are labelled. It appears that in spinach chloroplasts cp DNA is associated with granal thylakoids at intervals along the length of a continuous photosynthetic membrane system. Such an organization would facilitate cp DNA segregation during chloroplast division.

Chloroplasts↗
PubMed1984

Stochastic partitioning of chloroplasts at cell division in the alga Olisthodiscus, and compensating control of chloroplast replication.

We asked how chloroplasts in a unicellular marine alga are replicated and partitioned at cell division so that each daughter cell will receive the appropriate number of copies. The data were obtained simply by counting chloroplasts in pairs of daughter cells immediately after cell division. The results show that chloroplast partitioning is not always equal; however, it is equal much more often than predicted by the binomial distribution of chloroplast numbers that would be expected if partitioning were strictly random. The parental chloroplasts were partitioned equally in approximately 76% of the divisions, while in the remaining 24% the deviations from equality were very small. To maintain a reasonable range of chloroplast numbers in the face of unequal partitioning, there must be some form of compensating control of chloroplast replication. Our data suggest that daughter cells that receive very large numbers of chloroplasts go directly to the next division without replicating their chloroplasts, while cells with very small numbers of chloroplasts go through two rounds of chloroplast replication before dividing.

Cell Division↗
PubMed1983

Biosynthesis of chloroplast transfer RNA in a spinach chloroplast transcription system.

We have developed a chloroplast in vitro transcription system capable of transcribing tRNA genes (trn) from the spinach and Euglena gracilis chloroplast genomes. The RNA polymerase contained in the chloroplast extract transcribes the spinach chloroplast trnM2, trnV1, and trnl1 loci and the trnV1-trnN1-trnR1-trnL1 cluster in the EcoG fragment of the Euglena chloroplast genome. Restriction enzyme modified templates were used to demonstrate that the tRNA genes are transcribed in vitro. RNA fingerprint analysis confirmed that tRNAMetm, tRNAlle1 and tRNALeu are correctly processed transcripts from the spinach chloroplast trnM2, trnl1, and Euglena trnL1 loci respectively. CCAOH is added to the mature tRNAs in vitro by a 3' nucleotidyl transferase present in the chloroplast extract. Deletion mutants were constructed from the trnM2 locus to evaluate the role of 5' flanking sequences in transcription initiation and processing. DNA sequences between positions -56 to -85 upstream of the trnM2 locus are required for maximal transcription of tRNAMetm, but are not essential for processing. The RNA polymerase involved in chloroplast trn transcription is distinguishable from the RNA polymerase isolated as a DNA-protein complex from spinach chloroplast that is active in rRNA transcription.

Base Sequence↗
PubMed1969

Genetic functions of the chloroplast of Chlamydomonas reinhardi: effect of rifampin on chloroplast DNA-dependent RNA polymerase.

The effect of rifampin, an inhibitor of bacterial DNA-dependent RNA polymerase, was studied in Chlamydomonas reinhardi. It was shown, in vivo and in vitro, that chloroplast-located, but not nuclear, DNA-dependent RNA polymerase is inhibited by this drug. The inhibition of chloroplast RNA polymerase results in the inhibition of chloroplast rRNA synthesis, and thus in the loss of chloroplast ribosomes. The ability to carry out photosynthesis is also lost after prolonged heterotrophic growth in the presence of rifampin, but cell division and chloroplast replication are not affected. It is proposed that chloroplast DNA contains information for chloroplast rRNA, but this DNA does not have the information for chloroplast DNA polymerase. Moreover, the DNA polymerase is not synthesized on chloroplast ribosomes.

Chloroplasts↗
PubMed1988

Chloroplast rpoA, rpoB, and rpoC genes specify at least three components of a chloroplast DNA-dependent RNA polymerase active in tRNA and mRNA transcription.

The purpose of this study was to determine the relationship between putative chloroplast RNA polymerase subunit genes and known chloroplast transcriptional activities. We have prepared fusion polypeptide genes from fragments of chloroplast DNA homologous to bacterial RNA polymerase subunit genes and expression vectors carrying portions of the anthranilate synthetase gene (trpE). Fusion proteins for chloroplast homologs of the RNA polymerase alpha (rpoA), beta (rpoB), and beta' (rpoC) subunits were obtained from these genes. The fusion polypeptides synthesized by Escherichia coli in vivo were purified and used as antigens for production of rabbit polyclonal anti-RNA polymerase subunit-specific antibodies. The purified antibodies were able to immobilize chloroplast DNA-dependent RNA polymerases from spinach, pea, and Euglena gracilis. In addition, the soluble chloroplast RNA polymerase activity in tRNA and mRNA synthesis was strongly inhibited by these antibodies under conditions which had little effect on transcription by the chloroplast transcriptionally active chromosome that preferentially transcribed rRNA genes (Greenberg, B. M., Narita, J. O., DeLuca-Flaherty, C., Gruissem, W., Rushlow, K. A., and Hallick, R. B. (1984) J. Biol. Chem. 259: 14880-14887). From these data we conclude that the chloroplast genes homologous to bacterial RNA polymerase subunit genes are expressed in vivo and that the protein products specify at least three of the components of the chloroplast RNA polymerase(s) involved in tRNA and mRNA transcription.

Amino Acid Sequence↗
PubMed1980

Homology between chloroplast and prokaryotic initiator tRNA. Nucleotide sequence of spinach chloroplast methionine initiator tRNA.

The nucleotide sequence of a chloroplast methionine initiator tRNA from spinach has been determined. Although from a eukaryotic organism, this tRNA strongly resembles prokaryotic initiator tRNAs. Spinach chloroplast tRNAMetf has a much higher sequence homology with prokaryotic initiator tRNAs (81 to 84%) than with eukaryotic initiator tRNAs (64 to 69%). In addition, it possesses the two unique features of prokaryotic initiator tRNAs, lacking a base pair between the 5'-terminal residue and the fifth nucleotide from the 3'-end and containing a T-psi-C-A sequence in loop IV. Also, like prokaryotic initiator tRNAs, the chloroplast tRNAMetf is 77 nucleotides long and has few modified nucleosides (2'-O-methylguanosine, dihydrouridine, 7-methylguanosine, ribothymidine, and pseudouridine). This chloroplast initiator tRNA is strikingly different in sequence homology (55 to 62%), number of residues, and structure from mitochondrial initiator tRNAs. Restriction enzyme mapping techniques have shown that the chloroplast tRNAMEtf hybridizes to spinach chloroplast DNA. A set of characteristic chloroplast tRNA features seems to be emerging from a comparison of this tRNAMetf and several other chloroplast tRNAs which have been completely or partially sequenced. All have a 2'-O-methylated G-G sequence in the dihydrouridine loop, and the sequence T-psi-C-A, as opposed to T-psi-C-G, is predominantly found in loop IV. This is the reverse of the situation encountered in the overall non-chloroplast tRNA population.

Base Sequence↗