Biomedical subjects

J Ngernprasirtsiri

Publications and source records attributed to J Ngernprasirtsiri.

14 recordsLinked to original sources

PubMed1991

Characterization and Intraorganellar Distribution of Protein Kinases in Amyloplasts Isolated from Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Incubation of amyloplasts isolated from cultured cells of sycamore (Acer pseudoplatanus L.) with [gamma-(32)P]ATP resulted in the rapid phosphorylation (half-time of 40 seconds at 25 degrees Celcius) of organellar polypeptides. The preferred substrate for amyloplast protein kinases was Mg(2+). ATP, and recovery of only [(32)P]serine after partial acid hydrolysis indicated the predominance of protein serine kinases in the organelle. These activities were located in the envelope and stromal fractions of the plastid, which showed different specificities toward exogenous protein substrates and distinct patterns of phosphorylation of endogenous polypeptides. A 66-kilodalton polypeptide, inaccessible to an exogenously added protease, was one of the major phosphorylated products found in intact amyloplasts at low [gamma-(32)P] adenosine triphosphate concentrations. This polypeptide represented the major phosphoprotein observed with the isolated envelope fraction. The patterns of polypeptide phosphorylation found in intact amyloplasts and chloroplasts from cultured cell lines of sycamore were clearly distinguishable. The overall results indicate the presence of protein phosphorylation systems unique to this reserve plastid present in nonphotosynthetic tissues.

Journal Article↗

PubMed1990

Modulation of DNA methylation and gene expression in cultured sycamore cells treated by hypomethylating base analog.

The selective suppression of photosynthetic genes in both the nuclear and plastid genomes of the nonphotosynthetic white wild-type cell line of sycamore (Acer pseudoplatanus) has been found to be inversely related to the presence of a variety of methylated bases, especially 5-methylcytosine (5-MeCyt) and N6-methyladenine (N6-MeAde), localized in regions of the plastid genome containing silent genes. We used hypomethylating base analogs to manipulate the level of cytosine and adenine methylation in the white cells of sycamore, and examined the effects of changes in methylation on gene expression. Treatment with 5-azacytidine (5-AzaCyd) and N6-benzyladenine (N6-BzlAde) decreased cytosine and adenine methylation. This was accompanied by restoration of transcriptional activity in photosynthetic genes which are usually suppressed. Both 5-MeCyt and N6-MeAde suppressed nuclear gene expression, but only 5-MeCyt suppressed plastid gene expression.

Blotting, Northern↗

PubMed1990

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant. The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts.

Base Composition↗

PubMed1990

Application of an efficient strategy with a phage lambda vector for constructing a physical map of the amyloplast genome of sycamore (Acer pseudoplatanus).

Amyloplasts were isolated from a heterotrophic culture cell line of a woody plant, sycamore (Acer pseudoplatanus), and their DNA was purified. Conventional procedures for making a physical map were not easily applicable to the amyloplast DNA, since the yield of DNA was too low and the presence of repeated sequences interfered with the analysis. Therefore, the pieces of amyloplast DNA starting with a few micrograms of DNA were cloned in the lambda Fix vector, which is a derivative of lambda EMBL vectors improved for efficient cloning and gene walking. Cloned DNA fragments were randomly picked, mapped for restriction endonuclease sites by a refined procedure, and combined by overlapping their physical maps. The DNA library was also subjected to screening by gene walking using promoters recognized by T3 and T7 RNA polymerases in the vector to fill the gaps between sequences determined by overlapping the physical maps. In this way, we constructed the entire DNA library and the complete physical map of the amyloplast DNA. The sycamore amyloplast genome was composed of 141.7-kbp nucleotides with the same gene arrangement as that of tobacco chloroplasts.

Bacteriophage lambda↗

PubMed1990

Expression of photosynthetic genes is distinctly different between chloroplasts and amyloplasts in the liquid-cultured cells of sycamore (Acer pseudoplatanus L.).

A nonphotosynthetic, white-wild cell line of sycamore (Acer pseudoplatanus L.) contains amyloplasts as the only kind of plastid, whereas a photosynthetically competent green variant cell line contains only chloroplasts. Transcripts of both nuclear and plastid genes for photosynthetic components in the white cells were not detectable in contrast to those in the green cells. To investigate the limiting step (s) behind these diminished levels of transcripts, we have performed in vivo pulse-chase labeling of RNA in both cell types. These studies indicated that the rates of incorporation of [3H]uridine and nucleotide pool sizes were indistinguishable between the two cell lines. Transcripts of certain nuclear (rbcS, cab, psbO) and plastid (rbcL) genes in the white cell were not detectable. We infer from these data that transcriptional regulation entails an important role in controlling photosynthetic RNA levels. Related analyses exploiting plastid run-on transcription have provided supporting evidence that the transcription of the amyloplast genome in the white cell is greatly suppressed in contrast to that of the chloroplast genome in the green cell. The results support a model of selective suppression of photosynthesis genes in nonphotosynthetic higher plant cells, and indicate that gene expression in such a system is primarily controlled at the transcriptional level.

Cell Division↗

PubMed1990

DNA methylation is a determinative element of photosynthesis gene expression in amyloplasts from liquid-cultured cells of sycamore (Acer pseudoplatanus L.).

Transcriptional regulation has been shown to operate as a selective control mechanism of expression of photosynthetic genes in the nonphotosynthetic plastids, amyloplasts, of a white-wild cell line of sycamore (Acer pseudoplatanus L.). To elaborate the mechanisms governing the transcriptional regulation at the molecular level, we have examined the template activity of the amyloplast DNA compared to the chloroplast DNA by using the in vitro run-off transcription assay system with extracts of the two plastid types. The results of these assays clearly indicate that most of the amyloplast DNA regions do not serve as a template for the in vitro transcription regardless of the plastid extracts; this is in contrast to the chloroplast DNA which serves as an active template. It is highly likely that the template activity of amyloplast DNA per se is the modulating element of transcriptional regulation. Parallel experiments determining the DNA base content by HPLC analysis have shown that a variety of methylated bases, especially 5-methylcytosine, are localized in the DNA regions containing suppressed genes of the amyloplast genome. In sharp contrast, methylated bases were undetectable in the expressed gene regions of amyloplast and whole chloroplast genomes. The overall findings strongly support the notion that DNA methylation is involved in the selective suppression of photosynthetic genes in the nonphotosynthetic plastids of cultured sycamore cells.

Base Composition↗

PubMed1989

DNA methylation and the differential expression of C4 photosynthesis genes in mesophyll and bundle sheath cells of greening maize leaves.

The methylation of nuclear and chloroplast DNAs has been examined in relation to the known differential expression of C4 photosynthesis genes in the bundle sheath and mesophyll cells of etiolated, greening, and fully green maize leaves. We have focused our research on phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (RBUp2Case) which are coded by nuclear genes, and on the large subunit of RBUp2Case which is coded by a plastid gene. Reversed-phase high performance liquid chromatography revealed several kinds of methylated bases in DNAs of both photosynthetic cell types, with the largest amounts in fully green leaves. The occurrence of selective DNA methylation was investigated by employing an isoschizomeric pair of methyl-sensitive and -insensitive endonucleases followed by Southern hybridizations with specific DNA probes. Notably, there was an inverse correlation between the relative abundance of specific transcripts in a given cell type during greening and the methylation status of the corresponding nuclear or chloroplast gene. Furthermore, a heterologous in vitro transcription system using Escherichia coli RNA polymerase revealed that the plastid gene encoding the RBUp2Case large subunit in both cell types was active as a template in the unmethylated state, whereas it was inactive when methylated. Thus, the selective methylation of both chloroplast and nuclear DNA is likely one component of a multilevel control mechanism for the differential regulation of cell-specific C4 photosynthesis gene expression in greening maize leaves.

Cell Nucleus↗

PubMed1989

Transcriptional regulation and DNA methylation of nuclear genes for photosynthesis in nongreen plant cells.

The transcripts of nuclear genes for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcS), chlorophyll a/b-binding protein (cab), and extrinsic 33-kDa protein involved in photosystem II water oxidation (woxA) were not detectable in the white wild cultured cells of sycamore (Acer pseudoplatanus), in contrast to their high levels in the sibling green mutant cells and the constitutive expression of actin genes (act) in both cell types. We have examined the template activities of nuclear DNAs using the HeLa cell in vitro transcription system. All of the three photosynthesis genes from the green cell line and act from both cell types were well transcribed in vitro, but these photosynthesis genes from the white cell line were not, indicating that the transcriptional regulation is ascribable to DNA templates. Digestion of nuclear DNA with methyl-sensitive and -insensitive isoschizomeric endonucleases and the subsequent Southern hybridization showed that each gene has the identical recognition sites of restriction enzymes in the green and white cell lines, but some of the sites were methylated only in the photosynthesis genes in the white cells. There was observed a clear inverse relationship between the level of expressed transcripts and the extent of DNA methylation. Thus, it is inferred that the selective methylation of DNA is a likely mechanism for suppressing transcription of nuclear genes for photosynthesis in the nonphotosynthetic plant cells.

Base Composition↗

PubMed1989

Immunochemical Analysis Shows That an ATP/ADP-Translocator Is Associated with the Inner-Envelope Membranes of Amyloplasts from Acer pseudoplatanus L.

Pure preparations of intact amyloplasts and chloroplasts, free from mitochondrial contamination, were isolated from cultured cells of the white-wild and green-mutant lines of sycamore (Acer pseudoplatanus L.), respectively. A specific rabbit antiserum against yeast mitochondrial cytochrome c(1) only cross-reacted with mitochondrial membranes from the white-wild sycamore cells. The outer and inner envelope-membranes of the two plastid-types were isolated and subsequently analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis to characterize polypeptide patterns in each fraction. Analysis by immunoblotting clearly showed that antiserum against the 29-kilodalton inorganic orthophosphate translocator isolated from pea chloroplasts cross-reacted with a 31-kilodalton polypeptide residing in the inner-envelope membranes from both sycamore chloroplasts and amyloplasts. In contrast, antiserum against the ADP/ATP-translocator isolated from mitochondria of Neurospora crassa yielded a positive signal with a 32-kilodalton polypeptide in the inner-membranes isolated from amyloplasts, but not green-mutant chloroplasts. We propose that this 32-kilodalton polypeptide in the amyloplast envelope is a putative ATP/ADP-translocator and its possible functional significance is discussed.

Journal Article↗

PubMed1988

DNA methylation as a mechanism of transcriptional regulation in nonphotosynthetic plastids in plant cells.

Transcription of amyloplast DNA in a heterotrophic line of cultured cells of sycamore (Acer pseudoplatanus L.) appeared to be greatly suppressed. A mutant cell line obtained from the heterotrophic line is green and autotrophic. Heavy modification of amyloplast DNA with a variety of methylated bases was demonstrated by analysis of the acid hydrolysate of DNA by high-performance liquid chromatography, but little modification of chloroplast DNA from the green line was detected. When plastid DNAs from the original and green cell lines were digested with methyl-sensitive restriction enzymes, DNA methylation was detected in regions containing the genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL), subunits of chloroplast coupling factor 1 (atpA, -B, and -E), the apoprotein of P700 (psaA), and ribosomal protein S4 (rps4) but not the genes for 16S rRNA and the 32-kDa QB protein (psbA) in the original line, whereas no methylation was observed in the green line. The genes for which methylation was not detectable were found to be active as templates for in vitro transcription by Escherichia coli RNA polymerase, but the methylated genes were apparently inactive. Methylation of DNA is a likely mechanism for the regulation of expression of amyloplast DNA in sycamore cells.

Cells, Cultured↗

PubMed1988

Expression of Amyloplast and Chloroplast DNA in Suspension-Cultured Cells of Sycamore (Acer pseudoplatanus L.).

Green mutant cells of sycamore (Acer pseudoplatanus L.), which had been selected by mutagenic treatment of the white wild type, grow photoheterotrophically in auxin-depleted culture medium. In contrast to the wild-type cells, mutant cells exhibit photosynthetic O(2)-evolution activity during their growth coincident with increases of (a) chlorophyll, (b) protein, and (c) ribulose-1,5-bisphosphate (RuBP) carboxylase activity. Functionally competent chloroplasts were isolated from the green cells. Mechanism(s) governing gene expression of amyloplast DNA in the heterotrophically grown white cells were compared with those of the chloroplast DNA isolated from the mutant cells. We have demonstrated in both amyloplast and chloroplast DNAs the presence of sequences homologous to the maize chloroplast genes for photosynthesis, including the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)(rbcL), the 32 kDa Q(B) protein (PG32) (psbA), the apoprotein of P700 (psaA) and subunits of CF(1) (atpA, atpB, and atpE). However, employing either enzyme assays or immunological techniques, RuBisCO and CF(1) cannot be detected in the white wild type cells. Northern blot hybridization of the RNA from the white cells showed high levels of transcripts for the 16S rRNA gene and low level of transcripts for psbA; based on comparison with results obtained using the green mutant cells, we propose that the amyloplast genome is mostly inactive except for the 16S rRNA gene and psbA which is presumably regulated at the transcriptional level.

Journal Article↗

PubMed1988

Isolation and Characterization of the Amyloplast Envelope-Membrane from Cultured White-Wild Cells of Sycamore (Acer pseudoplatanus L.).

To study the characteristic features of the amyloplast, a uniquely differentiated plastid-type which synthesizes and accumulates reserve starch, in comparison with those of the chloroplast, these two types of plastids were isolated from white-wild and green-mutant protoplasts of cultured sycamore (Acer pseudoplatanus L.) cells, respectively. The intactness of the isolated amyloplast preparations was 70%. Electron microscopic ultrastructural analysis of both plastid types revealed unique structural features of the green-mutant chloroplasts, including well developed grana membranes and abundant ribosomal particles and plastoglobuli. After osmotic rupture of the isolated amyloplasts and chloroplasts, a clear separation of the envelope-membranes was achieved by discontinuous sucrose density gradient centrifugation. Although the visible absorption spectra of the envelope lipid components were indistinguishable between the amyloplasts and chloroplasts, the envelope-membrane polypeptide patterns were clearly distinct as judged by denaturing electrophoresis. By immunoblotting analysis using the specific antiserum raised against the pea chloroplast 29-kilodalton Pi-translocator, the amount of this carrier-protein (31-kilodalton) in the white-wild amyloplast envelope-membranes was estimated to be at least 10-fold less than in the green-mutant envelopes.

Journal Article↗

PubMed1988

DNA Methylation Occurred around Lowly Expressed Genes of Plastid DNA during Tomato Fruit Development.

We have analyzed DNA methylation of plastid DNA from fully ripened red fruits, green mature fruits, and green leaves of tomato (Lycopersicon esculentum var. Firstmore). Essentially identical restriction profiles were obtained between chromoplast and chloroplast DNAs by EcoRI digestion. BstNI/EcoRII and HpaII/MspI are pairs of isoschizomers that can discriminate between methylated and unmethylated DNAs. These endonucleases produced different restriction patterns of plastid DNAs from tomato fruits compared to tomato leaves. Moreover, we have found from Southern blots that methylation was not detected in DNA fragments containing certain genes that are actively expressed in chromoplasts, whereas DNA fragments bearing genes that are barely transcribed in chromoplasts are methylated.

Journal Article↗

PubMed1987

Sycamore amyloplasts can import and process precursors of nuclear encoded chloroplast proteins.

Amyloplasts isolated from white-wild suspension-cultured cells of sycamore (Acer pseudoplatanus L.) are found to import and process the precursor of the small subunit (pS) of ribulose-1,5-bisphosphate carboxylase/oxygenase of spinach, but they lack the ability to form its holoenzyme due to the absence of both the large subunit and its binding-protein. They also import the precursor of the 33-kDa extrinsic protein (p33-kDa) of the O2-evolving complex of Photosystem II from spinach, but process is only to an intermediate form (i33-kDa). Chloroplasts from green-mutant cells of sycamore process p33-kDa to its mature form in this heterologous system. These results suggest that the thylakoid-associated protease responsible for the second processing step of p33-kDa is missing in amyloplasts, possibly due to the absence of thylakoid-membranes. In contrast, the apparent import of the precursor of the light-harvesting chlorophyll a/b-binding apoprotein (pLHCP) from spinach was not detected. Sycamore amyloplasts may lack the ability to import this particular thylakoid-protein, or rapidly degrade the imported molecules in the absence of thylakoid-membranes for their proper insertion.

Chlorophyll↗